Durante o período de 2010 e 2011 dois experimentos foram conduzidos na Embrapa Clima Temperado, em Capão do Leão/RS. O primeiro envolveu a adição de citrato de sódio e bicarbonato de sódio na dieta de vacas Jersey, com o intuito de verificar influência destes aditivos no controle da incidência de LINA, como alegado por parte da(s) indústria(s) comercializadora (s) dos produtos. Dezessete vacas foram divididas em três tratamentos, em dois períodos experimentais com diferentes dietas. Grupo controle: 5 vacas sem receber aditivos na dieta; grupo Citrato: 6 vacas recebendo 100 g diárias de citrato de sódio; grupo Bicarbonato: 6 vacas com 40 g diárias de bicarbonato de sódio na dieta. Foram medidos os teores plasmáticos de glicose e ureia e o pH urinário, para monitoramento da fisiologia dos animais, assim como se determinou a composição físico-química do leite e a contagem de células somáticas. Não foi detectado efeito da adição de bicarbonato de sódio e citrato de sódio em nenhum dos parâmetros avaliados, demonstrando a ineficácia do uso destes aditivos no controle da incidência de LINA. Um segundo experimento, com duração de cinco semanas, também envolvendo vacas da raça Jersey, foi elaborado com o objetivo de determinar alterações na permeabilidade das tigh tjunctions das células epiteliais da glândula mamária provocadas por restrição alimentar severa. Doze vacas foram divididas em dois tratamentos contendo seis vacas cada: grupo Controle: animais recebendo dieta atendendo suas exigências nutricionais durante todo o experimento; grupo Restrição: animais recebendo dieta restrita em 50% somente durante a terceira semana. Determinou-se a permeabilidade das tight junctions através da mensuração dos níveis plasmáticos de lactose e dos teores lácteos de lactose e sódio. Foi determinada também a composição físico-química do leite e a contagem de células somáticas das amostras, assim como os teores de cortisol plasmático, para identificação da condição de estresse às quais os animais em restrição alimentar estavam submetidos. A restrição alimentar promoveu estresse aos animais do grupo Restrição, resultando em maior permeabilidade das tight junctions das células da glândula mamária, com efeitos sobre a redução da estabilidade do leite no teste do álcool. / In the period between 2010 and 2011 two experiments were conducted at Embrapa Clima Temperado, in Capão do Leão/RS. The first study involved the inclusion of sodium citrate and sodium bicarbonate in Jersey cow diet to determine the influence of this inclusion on the incidence of unstable non-acid milk (LINA). Seventeen animals were divided into three groups, in two experimental periods with different diets: Control group: 5 animals without the inclusion of additives in the diet; Citrate group: 6 animals receiving each 100 g of sodium citrate per day in the diet; Bicarbonate group: 6 animals receiving 40 g of sodium bicarbonate per day. Plasma concentration of glucose, urea as well as urine pH was measured to assess the animals’ physiologic status. Milk’s physical-chemical composition and somatic cell count were also determined. The inclusion of sodium citrate and sodium bicarbonate had no influence on any of the parameters evaluated, demonstrating the inefficiency of this technique in controlling the incidence of LINA. A second experiment, with five weeks duration also using Jersey cows, was carried out to define alterations in the permeability of mammary gland cells tight junctions due severe feeding restriction. Twelve animals were divided into two treatments with 6 cows each: Control group: animals receiving full diet during the entire study; Restriction group: animals receiving a 50% restricted diet only during the third week. Tight junction permeability was determined through plasma and milk levels of lactose and milk levels of sodium. Milk physical-chemical composition and somatic cell count were also determined. Cortisol concentration was measured to assess the animals stress condition. Feeding restriction induced stress in animals in the Restriction group, which promoted higher permeability of mammary gland cell tight junctions, with influences on the reduction of milk stability to the ethanol test.

Nous étudions la génération de rayonnement TeraHertz (THz) dans des microplasmas produits par des lasers femtosecondes. Cette technique est prometteuse pour créer efficacement des sources THz compactes et étendue spectralement (0.3-30 THz), qui intéressent de nombreuses applications, comme l’identification spectroscopique de substances dangereuses ou encore l’imagerie en biologie et médecine. Contrairement aux sources conventionnelles, comme les interrupteurs photo-conducteur, les sources THz basées sur des plasmas ne sont pas limitées par la tenue au flux et couvrent l’ensemble du spectre THz. Afin de modéliser des microplasmas générés par des faisceaux laser fortement focalisés, nous présentons un nouvel algorithme qui permet d’injecter tout type de laser dans des codes électromagnétiques. Nous dérivons aussi un modèle compatible avec les équations de Maxwell qui inclut les deux mécanismes générateurs de THz: le courant d’ionisation (IC) et le mécanisme “Transition-Cherenkov” (TC). Ce dernier mécanisme domine la production de THz pour des lasers à plusieurs cycles optiques, où l’émission est produite par les courants d’électron longitudinaux. Dans le cas des microplasmas où un champ électrostatique externe est ajouté, le taux de conversion énergétique laser/THz peut être augmenté de deux ordres de grandeur via le mécanisme IC lorsque le champs statique ou la pression du gaz sont accrus. De plus, les simulations 3D montrent que pour un faisceau laser à deux couleurs et dans des conditions optimales de focalisation, une énergie laser de 10 micro-Joule est suffisante pour atteindre des taux de conversion bien au-dessus de 10−4. Dans ce cas, la nature transverse du courant IC est cruciale pour accroitre l’efficacité avec la longueur du plasma. En considérant un faisceau laser à deux couleurs de forme elliptique, nous proposons de contrôler les spectres d’émission en exploitant les effets plasmoniques résonants. / We investigate terahertz (THz) generation in fs-laser-induced microplasmas, which are promising candidates for compact and efficient broadband THz sources (0.3-30 THz). Such sources have various applications as spectroscopic identification of hazardous substances or THz imaging in biology and medicine. Unlike conventional THz sources as photoconductive switches, gas-plasma-based THz sources do not suffer from irreversible material damage and can cover the whole THz range at once. To simulate tightly-focused-laser-induced microplasmas, we propose an efficient numerical algorithm that can introduce any arbitrarily shaped laser pulses into electromagnetic codes. We derive a Maxwell-consistent model that includes two major THz generation mechanisms, the ionization current (IC) and transition-Cherenkov mechanisms (TC). The latter mechanism is shown to dominate for single-color multi-cycle lasers pulses where the emission is driven by longitudinal electron currents. For microplasmas a constant electric field can boost the laser-to-THz converison efficiency by two orders of magnitude via the IC mechanism when increasing the gas-pressure and bias-voltage. Moreover for two-color-driving laser pulses, Maxwell-consistent 3D simulations show, that only 10 μJ laser pulse energy are sufficient to reach conversion efficiencies well above 10−4 when optimizing the focusing conditions. Here, the transverse nature of the IC currents is crucial for the up-scaling of the efficiency with the plasma length. By using elliptically-shaped two-color-driving laser beams, we propose to control the emission spectra by exploiting resonant plasmonic effects.

Abstract Acute pancreatitis is a common disease, Finland being among the countries with the highest incidence. The majority of patients have a mild, self-limiting disease. However, 20% of these patients develop severe necrotizing pancreatitis with a mortality rate of 7 to 25%. The mechanisms for developing the severe disease are not known, it is not possible to accurately forecast the severity of the disease and there is no curative treatment yet. This study was aimed at analyzing the early phase of acute experimental porcine oedematous and necrotizing pancreatitis. In Study I, the pancreatic microcirculatory changes were measured and the expression of tight junction proteins (claudins-2, -3, -4, -5 and -7) and the rate of apoptosis in the pancreas were all measured. In Study II, bacterial translocation to the blood in the portal vein blood or to the mesenteric lymph nodes was analyzed and the intestinal expression of tight junction proteins (claudins-2, -3, -4, -5 and -7) and the intestinal apoptosis/ proliferation rates were measured. The basic histology of the jejunum and colon were analyzed. Study III analyzed which cytokines are released from the pancreas to the portal venous blood. In Study IV, the ultrastructure of the epithelium of the jejunum and colon was analyzed and the expression of adherens junction proteins, E-cadherin and β-catenin, were measured from both jejunum and colon. The first study (I) showed that membranous immunoreactivity of claudin-2 in acinar cells appeared in the pancreas during acute oedematous and necrotizing pancreatitis. The expressions of claudins -3, - 4, - 5 and 7 were unaffected. The second study (II) showed that bacterial translocation from the gut was not present at the beginning of acute porcine pancreatitis. The expressions of claudins-2 and -5 do not become altered; however, there might be some decrease in claudin-3 expression in the colon and decrease in the expression of claudins-4 and -7 in the jejunum in necrotizing pancreatitis. Performing the laparotomy itself caused increased apoptosis in the colon and the jejunum. In the third study (III), the initial inflammatory process was diverse in oedematous and necrotizing pancreatitis. Increased monocyte count in combination with elevated PDGF and IL-6 are characteristic of necrotizing pancreatitis in our model. The fourth study (IV) indicated that necrotizing pancreatitis caused damage to the epithelial and endothelial cells of the colon in the early stages of the disease. The expression of E-cadherin immunoreactivity showed a decreasing trend in the colon in both oedematous and necrotizing pancreatitis. The results of this study suggest that claudin-2 increases in acinar cells during acute porcine pancreatitis. Bacterial translocation is not present during the early phase of acute porcine pancreatitis. Increased monocyte count and elevated PDGF and IL-6 are characteristic of early phase necrotizing porcine pancreatitis and necrotizing porcine pancreatitis causes damage to the epithelial and endothelial cells of the colon. / Tiivistelmä Akuutti haimatulehdus on yleinen sairaus, jonka ilmaantuvuus Suomessa on verrattain suuri. Suurimmalla osalla potilaista tauti on lievä ja itsestään paraneva. Kuitenkin 20 %:lle potilaista kehittyy vaikea haimatulehdus, johon liittyy 7–25 %:n kuolleisuus. On epäselvää, miksi toisinaan kehittyy vaikea tautimuoto. Taudin vaikeusastetta ei voida etukäteen tarkasti ennustaa, eikä tautiin ole parantavaa hoitoa. Väitöskirjatyön tarkoituksena oli tutkia lievän ja vaikean haimatulehduksen varhaisvaihetta kokeellisessa sikamallissa. Työssä I mitattiin haiman mikroverenkierron muutoksia, tutkittiin tiivisliitosproteiinien klaudiini-2:n, -3:n, -4:n, -5:n ja -7:n ilmenemistä sekä apoptoosin määrää haimassa. Toisessa työssä tutkittiin mahdollista bakteeritranslokaatiota porttilaskimovereen ja vatsaontelon imusolmukkeisiin, mitattiin suoliston tiivis liitos-proteiinien klaudiinien-2, -3, -4, -5 ja -7 ilmenemistä ja suoliston apoptoosin ja soluproliferaation määrää. Mahdollisia muutoksia ohut- ja paksusuolen perushistologiassa analysoitiin. Kolmannessa työssä mitattiin sytokiinipitoisuuksia porttilaskimoverestä. Neljännessä työssä analysoitiin ohut- ja paksusuolen mikrorakennetta elektronimikroskopian avulla ja mitattiin vyöliitosproteiinien E-cadherin ja β-catenin määrää. I työssä todettiin klaudiini-2:n ilmaantuvan haiman asinaarisolujen solukalvoille lievässä ja vaikeassa kokeellisessa haimatulehduksessa. Klaudiinien 3,- 4,- 5 ja 7 esiintyminen haimassa ei muuttunut. II työssä todettiin, että bakteeritranslokaatiota ei tapahtunut seuranta-aikana. Suolistossa klaudiinien-2 ja -5 ilmenemisessä ei tapahtunut muutoksia. Klaudiini-3:n ilmenemisessä paksusuolessa ja klaudiinien -4 ja -7 ilmenemisessä ohutsuolessa saattaa tapahtua vähenemistä vaikeassa haimatulehduksessa. Tutkimustoimenpide itsessään aiheutti ohut- ja paksusuolen apoptoosin lisääntymistä. III työn mukaan tulehdusvaste oli erilainen akuutissa lievässä ja vaikeassa kokeellisessa haimatulehduksessa. Monosyyttimäärän sekä PDGF:n ja IL-6:n pitoisuuksien lisääntyminen, olivat tyypillisiä vaikealle haimatulehdukselle tässä mallissa. IV työssä todettiin, että vaikea haimatulehdus vaurioittaa paksusuolen epiteeli- ja endoteelisoluja. E-cadherin: n määrässä todettiin jonkin verran vähentymistä sekä lievässä että vaikeassa haimatulehduksessa. Näiden tulosten mukaan klaudiini-2 lisääntyy sian haiman asinaarisoluissa akuutissa haimatulehduksessa. Sialla ei tapahdu bakteerien translokaatiota haimatulehduksen varhaisvaiheessa. Sian vaikeaan haimatulehdukseen liittyy monosyyttien, PDGF:n ja IL-6:n lisääntyminen. Kokeellisessa vaikeassa haimatulehduksessa paksusuolen epiteeli- ja endoteelisolut vaurioituvat jo varhaisvaiheessa.

No description available.

Abstract Squamous cell carcinoma (SCC) of the mobile tongue is the most common type of cancer of the oral cavity, accounting for 30-40% of oral cancers. It behaves aggressively and almost half of the affected patients still die of the disease despite great advances in its medical and surgical care. Ameloblastomas are the most common clinically significant type of odontogenic tumors, constituting approximately 1% of all cysts and tumors of the jaw. They are benign but locally invasive tumors with a strong tendency to recur after surgery. Ameloblastic carcinoma combines the histological features of ameloblastoma with cytologic atypia irrespective of the presence or absence of metastasis. The effectiveness of tight junction proteins (claudins 1, 4, 5, 7 and occludin) and cancer-associated fibroblasts (CAFs) as prognostic markers in OTSCC and as markers of malignancy in ameloblastomas was studied. Abundance of CAFs and Claudin 7 derangement was found to be associated with poor disease-specific survival in oral (mobile) tongue cancer. Appearance of CAFs within the epithelial islands of ameloblastoma was found to be a marker of malignancy in the tumor. The prognostic predictability of CAF density, Ki-67 (cell proliferation marker), maspin (tumor suppressor marker) and tumor DNA content (tumor ploidy using image cytometry) in tongue cancers was also tested. CAF density was the only marker strongly predictive of prognosis. In ameloblastomas, α-SMA (for CAFs), Ki-67, epithelial membrane antigen (EMA) and DNA content (using image and flow cytometry) were assessed as markers of ameloblastic carcinoma. Only α-SMA was able to predict ameloblastic carcinoma when found in the epithelial islands. In conclusion, staining for α-SMA and claudin 7 seems to be beneficial for prognostication in tongue cancer, while α-SMA staining may be beneficial in differentiating ameloblastoma from ameloblastic carcinoma.

Abstract Acute acalculous cholecystitis (AAC) is an insidious and increasingly recognized complication of critical illness, whose pathogenesis is poorly understood and clinical picture obscure. Diagnosis is difficult and there is no consensus on treatment. The medical records of all ICU patients who had undergone open cholecystectomy due to AAC during the years 2000–2001 and 2003–2004 were examined for clinical and organ failure data. The indication for open cholecystectomy was a suspicion of AAC based on clinical signs and symptoms of sepsis or deteriorating multiple organ dysfunction without other obvious foci and/or radiological (computed tomography or ultrasound) findings indicative of cholecystitis. A total of 73 patients had operatively treated AAC during the study periods, giving an incidence of 0.9% of all admissions (73/8184) and an incidence of 6.7% among the long-stayers (ICUstay >5 days). The hospital mortality of these patients was 43%. Infection was the most common admission diagnosis followed by cardiovascular surgery. The patients were severely ill, the mean (SD) APACHE II score being 25.5 (6.4) and the mean (SD) SOFA score 10.2 (3.5) on admission. In those patients who had AAC as the only intra-abdominal complication of multiple organ dysfunction, cholecystectomy was followed by a remarkable improvement of individual and total SOFA scores by the seventh postoperative day. The AAC gallbladders were histologically and immunohistologically compared to normal gallbladders and to gallbladders of patients with acute calculous cholecystitis (ACC). The ACC patients were admitted into hospital because of primary acute gallbladder disease, were treated on a normal ward and did not have severe sepsis or multiple organ dysfunction. The typical histopathological features of AAC (34 cases) in the gallbladder wall were bile infiltration, lymphatic dilatation and leucocyte margination of blood vessels, while epithelial degeneration and defects, widespread occurrence of inflammatory cells and extensive and deep muscle layer necrosis were typical features of ACC (28 cases). Tight junction proteins (claudin-1, -2, -3, -4, occludin, ZO-1 and E-cadherin) were uniformly expressed in normal gallbladder epithelium, with the exception of claudin-2, which was present in less than half of the cells. In AAC, the expression of cytoplasmic occludin and claudin-1 was decreased compared to control group. In ACC, the expression of claudin-2 was increased, but the expression of claudin-1, -3 and -4, occludin and ZO-1 was decreased compared to normal or AAC gallbladders. In conclusion, AAC is associated with severe illness, infection, long intensive care unit stay and deteriorating multiple organ dysfunction. Open cholecystectomy is one important contributing factor to reverse the course of multiple organ dysfunction in these patients. Histological and immunohistological studies suggest that AAC is a manifestation of systemic inflammatory disease, while ACC is a local inflammatory and often infectious disease.

Polymorphic light eruption (PLE) is the most common photodermatosis, affecting ~17% of the population. PLE is a delayed-type hypersensitivity response to an antigen induced by solar ultra-violet radiation (UVR). Its effects vary between patients, but the main symptom is a non-scarring, red papular rash in areas exposed to UVR. An effective therapy is low dose ultra-violet B (NBUVB) phototherapy. It is thought that NBUVB phototherapy desensitises the skin to further UVR exposure, but the mechanism by which this happens is unknown. Current immune based studies have been unable to clarify a mechanism as to how PLE arises. However, research in other skin diseases, such as psoriasis and atopic dermatitis, has shown that the barrier function of the skin is compromised by these disorders. Furthermore, research in lesional PLE skin showed an increase in barrier permeability of the skin. Recent research has specifically linked claudin proteins of tight junctions to the barrier dysfunction. Therefore, this study used quantitative immunofluorescent staining to measure tight junction (TJ) proteins and other barrier proteins of interest. Barrier function was also measured by transepidermal water loss (TEWL); a tracer dye penetration assay was used to measure TJ barrier function specifically. All measurements were made in non-lesional PLE skin, as compared to skin from healthy human volunteers. In photoprotected PLE skin the TJ protein claudin-1 was significantly reduced compared to healthy skin. The use of a tracer dye highlighted there was a reduction in TJ barrier function in PLE skin compared to healthy individuals. PLE and healthy skin were then exposed to ultra-violet B (UVB) and 24h later TJ proteins and TJ barrier function were measured. There was no change to claudin-1 after UVB exposure in PLE skin, but claudin-7 was reduced and claudin-12 increased. In contrast, in UVB-irradiated skin in normal controls after UVB exposure claudin-7 and claudin-12 were both increased, whilst claudin-1 was reduced. In PLE patients there was no further change to TJ barrier function, however, in normal controls, skin TJ barrier function was reduced post UVB. Both in healthy and PLE skin TEWL was unchanged before and after UVB exposure. Lastly TJ proteins were investigated after NBUVB in PLE patients. There was a further reduction in claudin-1 in PLE patients as well as a reduction in the TJ protein occludin, however the stratum corneum was significantly thickened. It could be suggested that this is a compensatory measure for the reduction seen in TJ barrier proteins, however further studies are needed to understand this. These data show significant differences in the TJ skin barrier in patients with PLE as compared to healthy human volunteers before and after UVB exposure. Furthermore, in PLE skin there is a significant change to the epidermis after NBUVB phototherapy. These data demonstrate that TJ protein expression and function is altered in PLE skin and may contribute to aetiology of the disorder, however the role of TJ barrier in aetiology is yet to be firmly established.

Doctor of Philosophy / Department of Biochemistry / Bruce D. Schultz / John M. Tomich / NC-1059 is a synthetic channel-forming peptide that provides for ion transport across, and transiently reduces barrier integrity of, cultured epithelial monolayers derived from canine kidney (MDCK cells; Broughman, J. R. et al; Am J Physiol Cell Physiol 286: C1312-23). In this first study experiments were conducted to determine whether epithelial cells derived from other sources were similarly affected. Human (T84, Calu-3) and non-human (IPEC-J2, PVD9902) epithelial cells derived from intestinal (T84, IPEC-J2), airway (Calu-3), and genitourinary (PVD9902) tissues were grown on permeable supports. Ion transport and barrier function were assessed electrically in a modified Ussing chamber. Basal short circuit current (I[subscript sc]) was typically less than 3 [Mu]A cm[superscript-2]. Apical NC-1059 exposure caused, in all cell types, an increase in I[subscript sc] to >15 [Mu]A cm[superscript-2], indicative of net anion secretion or cation absorption that was followed by an increase in transepithelial conductance (g[subscript te] in mS cm[superscript-2]; T-84, 1.6 to 62; PVD9902, 0.2 to 51; IPEC-J2, 0.3 to 26; Calu-3, 2.2 to 13). NC-1059 induces a concentration dependent change in the I[subscript sc] and g[subscript te] across these epithelia. The results in all cases were consistent with both a transcellular and a paracellular effect of the peptide. NC-1059 enhanced permeation of dextrans ranging from 10 kDa to 70 kDa across all epithelia tested. These results document an effect of NC-1059 on the paracellular route of epithelial barriers. Immunolabeling, confocal microscopy and immunoblotting methods were used in a second study to assess the molecular changes associated with increased paracellular permeability. NC-1059 induced a substantial reorganization of actin within 60 minutes of exposure. Confocal microscopy revealed that the changes in actin organization were accompanied by a pronounced change in the abundance and distribution of tight junction proteins occludin and ZO-1. Immunoblotting results suggest a time and concentration dependent effect on cellular abundance of these tight junction proteins. The effects on g[subscript te] and junctional proteins are transient with > 85% of recovery in 24 hours post exposure and full recovery within 48 hours. The reversible modulation of the epithelial tight junctions has therapeutic potential to increase the efficiency of drug delivery across barrier membranes.

The mammalian skin provides a necessary barrier between the organism and the environment, defending against loss of water and solutes, preventing the invasion of pathogens as well as protecting against chemical and physical assault. Claudin (Cldn)-based Tight Junctions (TJs) are the main functional part of the skin barrier. In particular, Cldn6 through its cytoplasmic tail has been shown to be important for barrier function. In other to further investigate the role of the Cldn6 tail in TJ-function, we developed Cldn6 mouse mutants carrying varying truncations of the Cldn6 tail. Both of these mice present with epidermal differentiation perturbations and delayed barrier function that is repaired later in life. These studies support the importance of the tail portion of the Cldn molecules in epidermal differentiation and barrier function. In addition, both of these mouse models are useful for the study of barrier function in preterm infants and in aging, with the hope of developing novel therapeutics for the alleviation of barrier dysfunction. Cdx is a family of homeodomain (HD) transcription factors (TFs) essential for many key developmental processes. In particular, Cdx2 is important for the establishment and maintenance of posterior identity in the developing endoderm. In spite of this, only a few Cdx targets in the developing endoderm have been discovered. In addition, the interplay between Cdx and its targets within the endoderm is poorly understood. In this study, we show that the forkhead box transcription factor, Foxa2 is a Cdx2 target. We also show that Foxa2 and Cdx2 physically and genetically interact to regulate a subset of genes that are implicated in endodermal development. These studies help to further our understanding of endoderm biology with the goal of developing new strategies to diagnose and treat diseases associated with defective endoderm development.

This thesis was created in cooperation with company Westfalia. The aim of this work is to analyze the possibility of rationalization of production of end parts for gas tight hoses used in the automotive industry. The thesis includes a study of rationalization of possible production of the second generation of end parts as well as completion of assembly line for the current generation of end parts.