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This study reports the localization and partial characterization of thioredoxin from the parasitic trematode Fasciola hepatica. Snails (Pseudosuccinia columella) were raised in culture and infected with F. hepatica so that Western blotting and immunohistochemical techniques could be utilized to determine the presence of thioredoxin in different stages of the parasite’s development. The results of these experiments showed that thioredoxin was present in the tegument, gut epithelium, excretory canal epithelium and sperm, of the adult parasite as well as in the tegument and gut of the redia and cercaria intermediate stages. In situ hybridization was used to determine the localization and possible differential mRNA expression of two different F. hepatica thioredoxin isotypes (Fh2020.A and Fh2020.SL) in the adult parasite. The in situ hybridization results showed that both isotypes are expressed in the tegument and gut epithelium. Fh2020.A stains with a greater intensity possibly demonstrating a difference in the amount of expression between the two isotypes. Recombinant F. hepatica thioredoxin expressed in bacteria using the pMAL™ Protein Fusion and Expression System was used to test its affects on the production of super oxide anion by murine peritoneal macrophages, bovine monocyte-derived macrophages and bovine whole blood neutrophils, and nitric oxide production by mouse peritoneal macrophages and bovine monocyte-derived macrophages. The results of the cellular assays were not definitive due to the fact that the maltose binding protein (MBP) moiety of the recombinant thioredoxin, when tested alone, increased production of nitric oxide by bovine monocyte-derived macrophages. Consequently, since the MBP could not be effectively separated from the thioredoxin portion of the recombinant, allowing the thioredoxin affects to be tested independently, no true conclusions regarding its affects on the host immune cells tested could be drawn. This is the first report of the localization of thioredoxin in both the adult F. hepatica as well as in specific intermediate stages of the parasite. These studies demonstrate the possible affects that a protein tag can have on experimental results and demonstrate how such data may be interpreted when a non-cleaved recombinant protein is used in cellular or other assays when compared to native or cleaved recombinant proteins.

Tez (Doktora) - Süleyman Demirel Üniversitesi, Fen Bilimleri Enstitüsü, Biyoloji Anabilim Dalı, 2009. / Kaynakça var.

Im Rahmen der Routinediagnostik von Endometriumbioptaten der Stute fielen regel-mäßig periglanduläre mononukleäre Entzündungszellinfiltrate (PAME) auf. Ziel der vorliegenden Studie war die histomorphologische und immunhistologische Charakterisierung der PAME im klinisch-gynäkologischen, jahreszeitlichen und endometrialen Kontext. Insbesondere sollte geklärt werden, ob das Phänomen der periglandulären Entzündungszellakkumulation Ausdruck eines physiologischen endometrialen Befundes, Frühstadium (Trigger) der Endometrose oder Begleitsymptom einer Endometritis ist. Zu diesem Zweck wurden alle im Jahr 2009 mittels einer Übersichtsfärbung (H.-E.-Färbung) im Institut für Veterinär-Pathologie der Universität Leipzig untersuchten Endometriumbioptate von Stuten (n = 754) hinsichtlich des Vorkommens einer PAME überprüft. Es konnten 133 Bioptate von 131 Stuten identifiziert werden, die ausführlich histopathologisch ausgewertet wurden. 72 Bioptate wurden zusätzlich anhand einer Methylgrün-Pyronin-Färbung (MGP) und immunhistologischer Verfahren beurteilt: Neben der Differenzierung der an der PAME beteiligten Zellpopulationen (CD3, CD79 A, MAC 387, MGP), wurde das Vorkommen der Intermediärfilamente Vimentin und Desmin sowie von α-GMA in den beteiligten Epithel- und Stromazellen untersucht. Die glanduläre Basallamina wurde anhand der Basallaminakomponente Laminin dargestellt und charakterisiert. PAME kamen in 18 % aller im Jahr 2009 untersuchten Bioptate vor, bevorzugt (96 %) in entzündlich und/oder fibrotisch alterierten Endometrien. Der Entzündungszellcharakter der begleitenden Endometritis stimmte in 29 % der Fälle nicht mit dem Entzündungszellbild der PAME (mononukleär) überein. Während im übrigen Endometrium häufig (95 %) eine gering- bis mittelgradige Endometrose nachweisbar war, wiesen die PAME-Drüsen mehrheitlich (71 %) keine periglanduläre Fibrose auf. Bei der Endometrose um Drüsen mit einer PAME handelt es sich überwiegend (67 %) um die destruierende und zu 50 % um die aktive/gemischte Form. In 48 % der PAME-Lokalisationen wurde eine Infiltration der periglandulären Entzündungszellen zwischen die Drüsenepithelzellen beobachtet. Die PAME bestanden hauptsächlich aus T-Lymphozyten (CD3-positiv). Daneben fanden sich, in geringerer und variierender Anzahl, Plasmazellen (MGP), B-Zellen (CD79A-positiv) und Makrophagen (MAC 387-positiv). Anhand der Expression der Basallaminakomponente Laminin wurden in allen untersuchten PAME-Lokalisationen Alterationen der Basallamina nachgewiesen. Die Läsionen der Basallamina waren bei mittel- und hochgradigen PAME graduell stärker ausgeprägt und standen häufig (43 %) mit einer intraepithelialen Infiltration der Entzündungszellen im Zusammenhang. Vimentin wurde vereinzelt (4 %) in intraläsionalen Drüsenepithelzellen nachgewiesen. Periläsionale Stromazellen exprimieren zu 60 % Vimentin, zu 17 % Desmin und zu 28 % α-GMA. Bei der PAME handelt es sich sehr wahrscheinlich nicht ausschließlich um ein „histopathologisches Symptom“ einer chronischen nicht-eitrigen Endometritis. Die Interpretation der PAME als physiologischer Bestandteil eines Schleimhaut-assoziierten lymphatischen Gewebes (MALT) im equinen Endometrium hingegen ist denkbar und bedarf in Folgeuntersuchungen der Klärung. Hinsichtlich der Strukturfilamente wurden in den involvierten Epithel- und Stromazellen immunhistologische Expressionsmuster nachgewiesen, die mit denen in frühen Stadien der equinen Endometrose vergleichbar sind. Darüber hinaus wiesen die periläsionalen Stromazellen Differenzierungsmerkmale von Myofibroblasten auf. PAME waren eng mit dem Vorliegen von Basallaminaalterationen verknüpft, die im Entstehungsprozess der equinen Endometrose eine zentrale Bedeutung besitzen. Es ist vorstellbar, dass es sich bei der PAME möglicherweise um einen auslösenden Faktor der Endometrose handelt. Außerdem ist eine intrinsische Aufrechterhaltung im Rahmen der zytokinvermittelten Interaktion (IL-4, IL-13, MCP-1), zwischen den periglandulären Entzündungszellen und den an einer Fibrose beteiligten Stromazellen, mit daraus resultierender progredienter Fibrose denkbar. Zukünftig sollte in weiteren Untersuchungen eine diesbezügliche Analyse der potentiellen Regelkreise erfolgen.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

There is a thermal range for the operation of neural circuits beyond which nervous system function is compromised. Poikilotherms are particularly vulnerable to thermal stress, since their body temperature can fluctuate with ambient temperature. Animals that experience frequent hyperthermia have various coping mechanisms such as the thermoprotective effect of a prior exposure to sublethal temperatures (heat shock response). The molecular mechanisms of this thermoprotection have yet to be understood. This project studies the changes in protein expression in the nervous system of gregarious Locusta migratoria subjected to heat shock. For this purpose, proteins were extracted from metathoracic ganglia (MTG) by different methods and a proteomic map was subsequently obtained by 2-D gel electrophoresis which was compared between control (CON) and heat-shocked (HS) animals. Additionally, the localization pattern of Hsp70 was studied in the MTG of CON and HS gregarious locusts. Although 2-D gels showed changes in the amount of different isoforms of ATP-synthase β, the overall amount of this protein subunit was found to be unchanged. My experiments also revealed no significant change in the distribution of Hsp70 in the MTG of locusts caused by HS. However, new findings show that this protein is constitutively expressed at higher levels in perineurium, glia and tracheal cells than in neurons. In separate experiments, isolated locusts were also examined in order to measure any stress-associated increase of Hsp70 in the tissues of animals not previously exposed to crowding pressure. Quantitative western blots did not show a consistent change of the Hsp70 level in the MTG of isolated locusts following heat shock. Results of my research suggest that the change in the protein profile of the metathoracic ganglion following heat shock, if it exists, is subtle or occurs in very low-abundance proteins whose monitoring requires the application of special techniques. Alternatively, the thermoprotective effect of heat shock on the nervous system might be promoted through other pathways which can change the protein activity at the post-translational level and may work independently from protein synthesis. / Thesis (Master, Biology) -- Queen's University, 2009-03-20 12:28:32.962

Most vertebrates suffer permanent damage after minutes of anoxia. Many insects however, have part of their life cycle in anoxia or constant hypoxia, such as during their egg-hatching phase, by living as deep burrowers, or at high altitudes. Insects are able to survive in anoxia from hours to days, or even months by developing various strategies through evolution. For example, the locusts (Locusta migratoria) enter a reversible coma during anoxia that is associated with an arrest of ventilation, and a reinitiation of ventilation when returned to normoxia. This coma is correlated with a surge in the concentration of extracellular potassium ions ([K+]o), and recovery from this reversible coma is dependent on re-establishing the functional [K+]o. Prior exposure to a sublethal heatshock (HS)-preconditioning grants locusts a temporary resilience to anoxia; however, the molecular mechanisms of this protection are still unclear. This project investigated the effects of HS-preconditioning on locusts’ ventilation, the total enzymatic activity of the Na+/K+-ATPase, as well as its distribution within the metathoracic ganglion and tested the hypothesis that HS-preconditioning alters locusts’ ventilation and increases the totally Na+/K+-ATPase activity and its concentration within neuronal membranes. I recorded electromyograms of locusts’ ventilatory motor patterns in the presence and absence of anoxic coma by placing a copper wire electrode on ventilatory muscles 161 or 173 in control and HS-preconditioned animals. In addition, I studied the enzymatic activity of the Na+/K+-ATPase using a pyruvate kinase/lactate dehydrogenase assay and the localization of the Na+/K+-ATPase using immunohistochemistry in control and HS-preconditioned locusts at different stages of coma. I found that the ventilatory cycle period was decreased and the ventilatory muscle burst duration was increased after recovery from anoxic coma in HS-preconditioned locusts. I also found that anoxia did not affect the activity or the localization of the Na+/K+-ATPase. However, HS-preconditioning increased the total activity of the Na+/K+-ATPase and the localization of the Na+/K+-ATPase within the neuronal membranes. From this project, I concluded that HSpreconditioning affected locusts’ ventilatory motor pattern after recover from anoxia and increased the total activity and the neuronal membrane localization of the Na+/K+-ATPase. / Thesis (Master, Biology) -- Queen's University, 2011-09-26 13:14:48.472

Infiltrative morphological mimicry at sites of biopsy-proven nodular basal cell carcinoma has been described. The immunoprofile of scar BCCs (scar BCCs,SBCCs) has not been documented. The aim of this study was to assess the histopathological spectrum, stromal (fibronectin, laminin, actin, desmin and vimentin) response and proliferation (bcl-2, MIB1 and p53) status of SBCCs. Twenty nine BCCs occurring in scars, unrelated to previous malignancy (de novo scar BCCS, DN-SBCCs), 27 BCCs that were incompletely excised and regrew at the same site (regrowth scar BCCs, RG-SBCCs) and 25 BCCs that were completely excised with tumour free margins, but recurred at the same site (recurrent scar BCCs, R-SBCCs) were accessed from the files of the Department of Pathology and Plastic and Reconstructive Surgery of the Faculty of Medicine, University of KwaZulu Natal, and formed the basis of this study. The morphological features of DN-SBCCs was pure (3%), predominantly nodular (79%), micronodular (7%) and infiltrative (11 %). RG-SBCCs were predominantly nodular (82%), micronodular (7%) and infiltrative (11%). RSBCCs were predominantly nodular (80%), micronodular (4%) and infiltrative (16%). The majority of DN-SBCCs, RG-SBCCs and R-SBCCs showed intact basement membrane laminin staining, while two (7%) DN-SBCCs showed 1 + and 2+ loss of basement membrane laminin staining. Three (11 %) and two (8%) RG-SBCCs and R-SBCCs,respectively, showed 2+ or 3+ basement membrane laminin discontinuity. The majority of DN-SBCCs (83%), RGSBCCs (75%) and R-SBCCs (88%) were actin negative. No desmin immunopositivity was demonstrated in the epithelial or stromal components of DN-SBCCs, RG-SBCCs and R-SBCCs. All BCC groups showed high 3+ or 4+ vimentin immunopositivity. The majority (>50%) of the SBCCs showed low (2+) bcl-2 immunopositivity. There was no significant difference in p53 immunopositivity in all SBCCs. SBCCs demonstrate phenotypic and immunophenotypic heterogeneity. That DN-SBCCs with the infiltrative and micronodular patterns have not recurred implies that the histomorphology is a pseudo-aggressive pattern. A similar view could pertain to RG-SBCCs, but because the scar did not cicatrise the incompletely excised BCC implies that the histomorphology of RG-BCC may be a potentially more aggressive phenotype. The recurrence of a completely excised basal cell carcinoma may be viewed as a feature of an aggressive tumour, especially when the recurrent BCC contains micronodular and infiltrative components. However, as most R-SBCCs occurred at head and neck sites that are exposed to ultraviolet light, it is also possible that these are simply new BCCs occurring within scars in head and neck sites prone to BCCs. Furthermore, these R-SBCCs were not destructive tumours. CONCLUSION: None of the infiltrative foci of DN-SBCCs demonstrated laminin loss. Three of 5 with intra-epithelial actin immunopositivity also demonstrated low bcl-2 and high p53 staining, immunoprofiling these with an aggressive infiltrative component. Of 11 RG-SBCCs with high p53 staining, 4 had high p53 staining in the infiltrative component, but only one had a low bcl-2 composite score and low bcl-2 score in the infiltrative focus. In addition, these infiltrative foci demonstrated intraepithelial MSA positivity and a "VA" immunophenotype of the stromal cells, indicating one RG-SBCC with an established, aggressive immunophenotype. Those positive with one or more, but not all, aggressive immunostains, are hypothesised to be RG-SBCCs evolving/developing an aggressive immunophenotype. Only one R-SBCC, with a predominantly infiltrative pattern, had a "full-house" of aggressive immunostaining in the infiltrative foci: low bcl-2, high p53, 2+ laminin discontinuity and intra-epithelial and stromal MSA positivity. Of significance is that 7 with a predominant nodular pattern had a high p53 score. Of these, 5 had high bcl-2 scores. Hence, while high p53 may be a feature of aggressive growth, it is important that this staining be complemented with that of bcl-2, laminin and MSA. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2006.

This series of experiments investigated dystrophin localization in the normal cerebellum and examined Purkinje neuron function in normal and dystrophin-deficient mice to better understand the physiological basis for cognitive deficits associated with Duchenne muscular dystrophy (DMD), a common genetic disorder among children. Cognitive impairments are consistently reported in DMD, yet precise mechanisms for their occurrence are unknown. Dystrophin protein, which is absent in DMD, is normally localized to muscles and specific neurons in the brain. Purkinje neurons are rich in dystrophin, specifically in somatic and dendritic membranes. Studies demonstrate perturbed cerebellar function in the absence of dystrophin, suggesting that DMD should be regarded as a cerebellar disorder in addition to being considered a neuromuscular disorder. However, theory and evidence are not generated from overlapping information: research investigating cerebellar involvement in DMD has focused on the vermal region, associated with motor function. The lateral region, implicated in cognition, has not been explicitly examined in DMD. The first experiment revisited the issue of dystrophin distribution in the mouse cerebellum using immunohistochemistry to investigate qualitative and quantitative differences between cerebellar regions. Both regions showed dystrophin localized to Purkinje neuron somatic and dendritic membranes, but dystrophin density was 30% greater in the lateral than the vermal region. The second experiment examined intrinsic electrophysiological properties of vermal and lateral Purkinje neurons from wild-type (WT) mice and from the mdx mouse model of DMD which lack dystrophin. Significant differences in action potential firing frequency, regularity, and shape were found between cerebellar regions. Purkinje neurons from mdx mouse cerebellum exhibited membrane hyperpolarization and irregular action potential firing, regardless of region. Spontaneous action potential firing frequency was reduced in Purkinje neurons from lateral cerebellum in mdx mice relative to controls, demonstrating that a loss of dystrophin causes a potent dysregulation of Purkinje neuron function in the region associated with cognition. This research extends our understanding of cerebellar pathology in DMD and its potential relevance to cognitive deficits in the disorder. Moreover, this research further supports the role of the cerebellum as a structure important for cognition and contributes to our understanding of dystrophin’s role in the brain.

The 12th International St Gallen conference on breast cancer (2011) proposed patient categorization for systemic therapy, based on intrinsic breast cancer subtypes, defined by imunohistochemistry (IHC) test results. Since this classification is based on semi-quantitative expression of IHC biomarker expression, an issue of defining and applying cutoff values remains. Essential improvement in the IHC testing has become possible with digital image analysis tools enabling quantitative evaluation of the IHC data. This study explores data obtained by digital image analysis methods applied to evaluate a comprehensive biomarker dataset (p53, AR, p16, BCL2, SATB1, HIF1) along with well established (ER, PR, HER2, Ki67) biomarkers. Also, an extensive set of genetic and epigenetic biomarkers has been tested. For the first time, the dataset of 10 IHC biomarkers, evaluated by digital analysis was explored by the means of factor analysis to establish the intrinsic factors of biological variation and informative value of IHC biomarkers and their combinatiions. The results also provided insights into the significance and combinatorial effects of the established and relatively new biomarkers (p16, SATB1, HIF1, Ki67/BCL2, etc.). / XII tarptautinėje St Gallen krūties vėžio konferencijoje (2011) Ekspertų komisijos priimta nauja pacientų klasifikacija sisteminei terapijai atlikti, paremta biologiniais krūties vėžio subtipais, kurie apibrėžiami imunohistocheminiu tyrimu (IHC). Tačiau ši nauja navikų klasifikacija iš esmės pagrįsta pusiau kiekybiniu biožymenų raiškos vertinimu, todėl išlieka aktuali ribinių verčių nustatymo problematika. Esminiai pokyčiai IHC tyrimų srityje galimi atsiradus skaitmeninio vaizdinimo technologijoms, leidžiančiomis IHC tyrimų rezultatus analizuoti kiekybiniais parametrais. Darbe naudojant skaitmeninį vaizdo analizės metodą atliktas išsamus biologinių žymenų tyrimas leido palyginti svarbių, tačiau nepakankamai ištirtų (p53, AR, p16, BCL2, SATB1, HIF1) IHC žymenų informatyvumą su esamų prognozinių žymenų (ER, PR, HER2, Ki67) rodikliais. Ištirtas platus genetinių ir epigenetinių krūties vėžio žymenų spektras. Pirmą kartą 10 IHC žymenų rinkinio, įvertinto skaitmeninės analizės būdu, duomenys panaudoti faktorinės analizės metodu nustatyti jų variacijų vidinius veiksnius, atskleidžiančius biologinius dėsningumus ir IHC žymenų bei jų derinių informatyvumą. Šios analizės rezultatai leido naujai įvertinti publikuotų krūties vėžio IHC žymenų bei jų derinių (p16, SATB1, HIF1, Ki67/BCL2 ir kt.) informatyvumą.