As cascavéis são classificadas como grupo monofilético contendo dois gêneros descritos ao grupo: Crotalus ssp. e Sistrurus ssp., os quais surgiram no México a aproximadamente 20 milhões de anos, colonizando então, praticamente todo o continente americano. Fatores como dieta, dimorfismo sexual, ontogenia, mutações e distribuição geográfica podem influenciar na composição dos venenos e consequentemente no envenenamento. O presente trabalho tem como objetivo caracterizar o perfil proteico, bem como as propriedades enzimáticas e imunológicas dos venenos de algumas espécies e subespécies de Crotalus ssp. (C. atrox, C. scutulatus scutulatus, C. viridis viridis, C. vegrandis, C. durissus cascavella, C. d. collilineatus e C. d. terrificus). Os resultados indicaram pouca variabilidade entre os perfis eletroforéticos dos venenos, contudo as diferenças foram na concentração relativa das proteínas. A análises proteômica identificou alguns componentes dos venenos e serinopeptidases, metalopeptidases e fosfolipases A2 foram as mais abundantes. Além disso, por zimografia, observou-se que todos os venenos analisados apresentaram atividade proteolítica e que os venenos norte-americanos em todos os zimogramas foram mais hidrolíticos. Em caseína, a atividade enzimática dos venenos foi menos intensa comparado aos outros substratos. Em relação às gelatinases das amostras estudadas, pôde ser observado inibição da atividade enzimática induzida por alguns componentes utilizando EDTA, principalmente nos venenos de C. atrox e C. vegrandis. Em relação à inibição das serinopeptidases, foi observado que todas as gelatinases dos venenos crotálicos apresentaram inibição total ou parcial da atividade hidrolítica. Houve variabilidade entre as hialuronidases encontradas dos venenos crotálicos, tanto em relação à massa das enzimas e intensidade da degradação, quanto em diferentes pHs. Nos ensaios enzimáticos quantitativos (azocaseinolítico fosfolipásico e peptidásico) os venenos Norte Americanos demonstraram conter mais proteases em relação aos venenos Sul Americanos. Por Western Blotting, as amostras reagiram com os anticorpos presentes nos soros anti-crotálico e anti-botrópico, apresentando reatividade antigênica cruzada entre as amostras homólogas e heterólogas. Além disso, houve imunoreatividade entre o soro anti-jararagina e alguns componentes de todos os venenos crotálicos norte-americanos. / The rattlesnakes are classified as a monophyletic group containing two genera referring to the group: Crotalus ssp. and Sistrurus ssp., which arose in Mexico 20 millions of years ago, colonizing then, practically all the American continent. Some scientific works indicate that factors such as diet, sexual dimorphism, ontogeny, mutations and distribution may influence the composition of the venoms and consequently the poisoning. The present work aims to characterize the enzymatic and immunological properties of the venoms of some species and subspecies of Crotalus ssp. (C. atrox, C. scutulatus scutulatus, C. viridis viridis, C. vegrandis, C. durissus cascavella, C. collilineatus and C. d. terrificus). The results indicated few variability among the electrophoretic profiles of the venoms, however the differences were in the relative concentration of the proteins. The proteomic analysis identified serinopeptidases, metallopeptidases and phospholipases A2, which were the most abundant components of the venoms. In addition, zymography assays indicate that the all the venoms showed proteolytic activity, furthermore, the North American venoms, presented more hydrolysis in all zimograms. The caseinolytic activity was less intense compared with other substrates. Regarding the gelatinolytic activity of the samples, inhibition of the enzymatic activity of some components could be observed using EDTA, mainly in the C. atrox and C. vegrandis venoms. Partial or total inhibition was observed of the serinopeptidases activity of the crotalic gelatinases. Among the hyaluronidases, variations between crotalic venoms, in relation to the enzymes mass and degradation intensity were identified. In addition, when incubated at different pHs, the hyaluronidase profile presented different patterns in the activity. In the quantitative enzymatic assays (azocaseinolytic phospholipasic, peptidasic) the North American venoms displayed higher activity in relation to the South American venoms. In the Western Blotting assays, the samples reacted with antibodies present in the Brazilian anti-crotalic and bothropic sera, indicating cross-reactive antigenicity between the homologous and heterologous samples. Besides that, there was immunoreactivity between the anti-jararrhagin serum and some components of all North American crotalic venoms.

The effect of iron absorption on subsequent hemoglobin regeneration was investigated by repletion and subsequent analysis of an anemic weanling rat model. Primary assay procedure in these studies was the Regeneration Efficiency method. As repletion or curative assay techniques form the central body of investigative research in iron utilization, the first subject of inquiry was the effect of repletion interval and degree of depletion upon the animals as monitored by physical indicators such as blood volume, growth and hematinic response. Percent blood volume is a particularly useful figure in the calculation of the amount of hemoglobin iron but was found to be relatively constant in a preliminary experiment. Accordingly, the effects of growth and anemia on hemoglobin response and blood volume were examined in 122 weanling male rats which had been depleted for seven days by low iron diet and phlebotomy, then repleted by feeding iron rich diet (47.1 ppm) for zero, five, 10 and 15 days. Percent blood volume proved to be rather constant at 7.5 in these pre-pubescent animals. The most severely depleted animals appeared to regenerate hemoglobin initially rather than replenish tissue iron. Regeneration Efficiency and AOAC assay methods were compared in a study involving the relative potency of two iron salts, ferrous sulfate and ferric orthophosphate, and three commercial cereal sources containing electrolytically reduced, hydrogen reduced or ferric orthophosphate supplements. These two assays utilized 202 albino male weanling rats. The superior availability of ferrous sulfate was evident in both regeneration efficiency and AOAC assays, as was the relative similarity of the dietary iron sources, yet the commercial sources in general provided more available iron in comparison to the reference ferrous sulfate than expected, possibly due to the influence of dietary components and processing variables. Bioavailability based on dietary iron concentration response correlated highly (r = 0.94) between animal groups analyzed using the Regeneration Efficiency and AOAC methods. Stress may have been a factor in animals assayed by the AOAC technique which used a 28 day depletion interval followed by a two week repletion. The Regeneration Efficiency method, which utilizes shorter depletion and repletion intervals also offered sufficient amounts of dietary iron for normal growth. The calculation of iron bioavailability also is dependent on such pertinent variables as dietary intake amount, body weight gain and percent blood volume.

Abnormal T lymphocyte reactions in both autism and Alzheimer's disease (AD) have been reported. This research investigated the possibility that these abnormalities may involve circulating antithymic antibodies. Plasma samples from autistic patients, AD patients, and normal-matched controls were tested for reactivity against murine thymocytes. In the first of 3 studies results of the enzyme-linked immunosorbent assay (ELISA) were statistically significant for binding (P < 0.001) between antithymic antibodies in plasmas of AD patients and murine thymocytes. Binding (P < 0.05) in low dilutions (1/2.5 and 1/5} of autistic patient plasmas was also observed. In the second study, plasmas of neither autistic nor AD patients significantly inhibited DNA synthesis of thymic cells in the presence of interleukin-1 (IL-l} and phytohemagglutinin (PHA). In the third study, no significant increases (P > 0.05) in cytotoxic activities were detected using AD patient plasmas and both untreated and heat-treated autistic patient plasmas. After further testing, these heat-treated plasmas diluted 1/64 and 1/128 had increased cytotoxicities (P Therefore, circulating antithymic antibodies may be involved in abnormal T lymphocyte reactions in autism and AD. Since they probably do not act alone, future research should study these complex abnormalities using human thymocytes.

FAPα ist ein Membranglykoprotein mit Dipeptidyl-Peptidase- und Typ I Kollagenase-Aktivität, das eine wichtige Rolle im Rahmen des Gewebeumbaus und der Angiogenese spielt. Die Expression von FAPα wurde für eine Reihe von Geweben gezeigt. So konnte bereits gezeigt werden, dass FAPα nach Myokardinfarkt auf humanen kardialen Fibroblasten (HCF) verstärkt exprimiert wird. Ausgehend von der Erkenntnis, dass eine im Blut zirkulierende Form von FAPα, APCE, gefunden wurde, sollte in der vorliegenden Arbeit eine mögliche Assoziation des in HCF exprimierten FAPα mit dem zirkulierenden APCE untersucht werden. Hierfür wurden ebenfalls die Signalwege, die zur Induktion von FAPα führen, näher untersucht. Im Rahmen der vorliegenden Arbeit konnte gezeigt werden, dass das von humanen kardialen Fibroblasten exprimierte FAPα ebenfalls im Zellkulturüberstand nachgewiesen werden kann. Wie bereits beschrieben, führte eine Stimulation mit TGFβ1 zur Expression von FAPα. Diese ist abhängig von Smad-3 und konnte durch Zugabe des Inhibitors SB431542 blockiert werden. Auch Smad-2 scheint die FAPα-Expression zu beeinflussen. Ein Effekt von EGR-1, CTGF und TNFα auf die FAPα-Expression konnte hingegen nicht nachgewiesen werden. Darüber hinaus wurde im Rahmen der vorliegenden Arbeit ein ELISA zur Messung von humanem FAPα im Plasma erstmalig etabliert. In der Evaluation des ELISA wurden die Grenzwerte für die Detektion und Quantifizierung bestimmt, die Intra- und Intervariabilität des ELISAs berechnet und die Linearität der Wiederfindung von humanem rekombinantem FAPα bewertet, sowie die Stabilität von FAPα nach mehrfachen Einfrier-Auftau-Zyklen und nach der Lagerung bei verschiedenen Temperaturen evaluiert. Da hier nur kommerziell erwerbliche Materialien verwendet wurden, ist eine Anwendung durch Dritte leicht durchführbar. Es wurde weiterhin eine klinische Studie zur Messung der FAPα-Plasmaspiegel in 101 gesunden Blutspendern und 407 Patienten mit akutem Koronarsyndrom (ACS) sowie von 25 Patienten mit stabiler koronarer Herzerkrankung (KHK) durchgeführt. Hierbei konnten erstmals Referenzwerte für zirkulierendes FAPα im Plasma beschrieben werden. Die FAPα-Plasmaspiegel der gesunden Kontrollgruppe unterschieden sich nicht von denen der Patienten mit stabiler KHK. Bei Patienten mit akutem Koronarsyndrom war die FAPα-Konzentration im Vergleich mit den FAPα-Plasmaspiegeln der Kontrollgruppe hingegen signifikant erniedrigt. Auch zeigte sich bei den Patienten mit FAPα-Spiegeln im kleinsten Quartil eine erhöhte Mortalität. Die biologische Funktion von FAPα bzw. dessen mögliche Pathophysiologie im Rahmen eines ACS sowie die mit niedrigen FAPα-Spiegeln assoziierte Mortalität sind noch unklar und bleiben Gegenstand der weiteren Forschung. / FAPα is a membrane protein with dipeptidyl-peptidase and type-I-collagenase activity involved in angiogenesis and tissue repair. A number of cells and tissues including human cardiac fibroblasts (HCF) after myocardial infarction express FAPα. Due to the recent discovery of a circulating form of FAPα, APCE, a possible association between the cell-bound form of FAPα and APCE was studied in HCF. Furthermore, the signaling pathways that lead to an induction of FAPα were analyzed. It could be demonstrated that HCF shed FAPα, which can be detected in the cell culture supernatant. Moreover, in accordance with earlier results, FAPα expression is induced by TGFβ1. This is dependent on Smad-3 and could be blocked by the inhibitor SB431542. Additionally, Smad-2, but not EGR-1, CTGF or TNFα are involved in FAPα expression. Within this work an ELISA for the quantification of FAPα in human plasma was established and the limits of detection and quantification, the intra- and interassay variability and the linearity of recovery of human FAPα were determined. Furthermore, the stability of FAPα after multiple thaw-freeze-cycles or different storage conditions was evaluated. Because of the exclusive use of commercially available products, others could easily repeat this ELISA. FAPα plasma concentrations in 101 healthy blood donors, 407 patients with acute coronary syndrome (ACS) and in 25 patients with stable coronary artery disease were quantified by ELISA. This is the first time reference values for FAPα plasma levels could be derived. There was no significant difference between healthy donors and patients with stable coronary artery disease. However, compared to the healthy control group, FAPα levels were significantly lower in patients with ACS. Importantly, ACS patients with FAPα levels in the lowest quartile showed an increased mortality. The biological function of FAPα and its possible pathophysiological role in ACS are still unclear and remain the object of future studies.

Stammzellbasierte Therapieverfahren versprechen neue Lösungen für bisher nur unzureichend behandelbare Erkrankungen. In der Hals-, Nasen- und Ohrenheilkunde ist die Herstellung von Knorpel im Rahmen des Tissue Engineering von besonderem Interesse. Die mesenchymalen Stammzellen des Fettgewebes (ASC) stellen eine vielversprechende Zellpopulation als Ausgangspunkt für die Erzeugung von Gewebe dar. Auf Grund der hohen Zahl an Zellteilungen, oxidativem und mechanischem Stress sowie enzymatischer Verdauung steigt im Rahmen der in vitro Expansion das Risiko für DNA-Schäden. Diese können wiederum der Ausgangspunkt für die maligne Transformation einer Zelle sein. Ziel unserer Studie war es, zu zeigen, ob die Expansion und mehrfache Passagierung zu einer zunehmenden genetischen Instabilität der ASC führt. Es wurden frische ASC aus Liposuktionsaspirat von 8 verschiedenen Patienten isoliert. Mit ASC der Passagen 1, 2, 3, 5 und 10 wurde zur Detektion von Schäden auf DNA-Ebene jeweils eine alkalische Einzelzellgelelektrophorese(Comet Assay) und ein Mikrokerntest durchgeführt. Zur Erfassung von Schäden auf Chromatidebene erfolgte darüber hinaus mit Zellen der selben Passage ein Chromosomenaberrationstest. Mit dem Comet Assay und dem Mikrokerntest konnte keine signifikante Progression der genetischen Instabilität mit zunehmender Passage nachgewiesen werden. Beim Chromosomenaberrationstest zeigte sich im Friedman-Test eine signifikante Zunahme an strukturellen Chromosomenaberrationen mit steigender Passage. Der Wilcoxon-Test hingegen erbrachte kein signifikantes Ergebnis. Die im Rahmen dieser Arbeit gewonnen Daten zeigen, dass eine zunehmende genetische Instabilität der ASC mit zunehmender Dauer der Expansion und steigender Passage nicht vollständig ausgeschlossen werden kann. Aus diesem Grund sollten vor einer Transplantation regelhaft Untersuchungen wie beispielsweise ein Chromosomenaberrationstest oder ein Screening auf typische malignitätsfördernde Mutationen erfolgen. / Stem-cell based therapies promise new solutions for diseases which are insufficiently treatable up to now. In Otorhinolaryngology, the in vitro production of cartilage for tissue engineering approaches is of particular interest. Mesenchymal adipose-derived stem cells (ASCs) are a promising cell population for the production of tissue. Due to a high number of cell divisions, oxidative and mechanical stress as well as enzymatic digestion there is an increasing risk of DNA-damage during in vitro expansion. This DNA-damage can lead to a malignant transformation of the ASCs. The aim of our study was to show whether prolonged in vitro expansion leads to an increased genetic instability of ASCs. Human ASCs were isolated from subcutaneous adipose tissue of different donors (n = 8) undergoing liposuction surgery for aesthetic reasons. To detect DNA-damage, an alkaline single-cell microgel electrophoresis (comet) assay and a micronucleus assay were performed with cells of passage 1,2,3,5 and 10. Moreover, to assess chromosomal damage, a chromosomal aberration test was carried out with cells of the same passage. With the comet assay and the micronucleus assay, no significant progress of DNA-damage could be demonstrated. However, the chromosomal aberration test showed a significant increase of structural chromosomal damage. The results of our study underline the fact that an increasing genetic instability of ASCs during prolonged in vitro expansion cannot be completely excluded. Consequently, tests monitoring malignant transformations or genetic instability should be implemented before transplantation of ASCs.

Hintergrund: Die Studienlage zu Kautabak und Zigarettenrauch ist eindeutig und zeigt karzinogenes Potential. Über Schnupftabak ist hingegen wenig bekannt, vor allem auf zellulärer Ebene gibt es keine ausreichenden wissenschaftlichen Publikationen. Somit lässt sich die eventuell mutagene Wirkung von Schnupftabak nur schwer einschätzen. In Konsequenz stützt sich die WHO in ihrer Einstufung des Schnupftabaks als nicht karzinogen auf eine sehr eingeschränkte Datenlage. Ziel: Ziel der vorliegenden Arbeit war es, Schnupftabak auf mögliche zyto- und genotoxische Effekte auf humane Lymphozyten und Nasenschleimhautzellen zu untersuchen um ggf. tumorinitiierende Effekte darzustellen. Material und Methoden: Es kam eine Schnupftabaksorte ohne Menthol und eine Sorte mit Mentholzusatz zum EInsatz. Die benötigten Nasenschleimhautzellen und Lymphozyten wurden von 10 Probanden gewonnen und eine Stunde lang mit einem Schnupftabak-DMSO-Gemisch (2000µg/ml bis 0,01µg/ml) inkubiert. Zur Analyse wurde der Trypanblautest, dee Comet Assay und der Mikrokerntest verwendet. Ergebnis: Der Trypanblautest zeigte keinen Abfall der Vitalität. Beim Comet Assay ergab sich bei Lymphozyten ein signifikanter Anstieg der DNA-Fragmentierung ab 100µg/ml, bei Nasenschleimhautzellen ab 1000µg/ml. Der Mikrokerntest wies keine signifikante Zunahme der Mikrokerne auf. Es konnte kein Unterschied zwischen den beiden Tabaksorten aufgezeigt werden. Diskussion: Es zeigte sich eine Schädigung der Erbsubstanz im Comet Assay, die möglicherweise reparabel ist. Irreparable DNA-Schäden im Sinne von Mikrokernen wurden nicht gefunden. Nach diesen Ergebnissen muss die Einstufung der WHO in Zweifel gezogen werden. Untersuchungen mit weiteren Endpunkten der Genotoxizität sind somit gerechtfertigt, um zu einer fundierten Beurteilung des Risikopotentials von Schnupftabak zu gelangen. / Background: While an abundant number of studies concerning tobacco smoke and chewing tobacco show carcinogenic potential, there is little data on the consequences of snuff, especially on the cellular level. Therefore, the mutagenic effect of snuff is hard to estimate and the WHO assessment of snuff being not carcinogenic bases on very limited data. Objectives: This paper investigates potential cytotoxic and genotoxic effects of snuff on human lymphocytes and nasal mucosa cells. Materials and methods: Two kinds of snuff were used, one with a high degree of essential oil. The necessary nasal mucosa cells and lymphocytes were taken from 10 subjects undergoing nasal obstruction surgery and incubated with a snuff mixture (from 0,01µg/ml to 2000µg/ml). Methods included the trypan blue test, the comet assay and the micronucleus test. Results: The trypan blue test showed no decrease in cell viability for both cell types. The comet assay revealed a significant increase in the Olive Tail Moment for lymphocytes starting at 100µg/ml and 1000µg/ml for nasal mucosa cells. There was no significant increase in micronuclei according to the micronucleus test. Conclusion: The present study demonstrated genotoxic damage, such as DNA strand breaks, which may be repaired, but no non-repairable elevated micronuclei. The present findings cast doubts the WHO assessment that snuff is not carcinogenic, however, further research on various genotoxic endpoints in human cells are warranted.

Lifestyle diseases represent a large burden on developed societies and account for much morbidity worldwide. Research has shown that eating a diet rich in fruit and vegetables helps to ameliorate and prevent some of these diseases. Antioxidants found in fruits and vegetables may provide a substantial benefit in reducing disease incidence. This thesis examines the antioxidant properties of resveratrol, piceatannol, and pterostilbene, and the ability of Burkitt's Lymphoma (Raji) cells to uptake these three antioxidants. It also studies the effect of the antioxidants in protecting against DNA damage and their role in DNA repair following oxygen radical exposure in Raji cells. The Oxygen Radical Absorbance Capacity (ORAC) assay was used to measure overall antioxidant contribution as well as the ability of Raji cells to uptake antioxidant following exposure to 2,2’-Azobis(2-methyl-propionamide) dihydrochloride (AAPH). The single cell gel electrophoresis (Comet) assay was used to assess DNA damage and DNA repair rates of cells. Results showed that Raji cells, following oxygen radical exposure, significantly uptake pterostilbene (p < 0.0001), but not piceatannol or resveratrol. Piceatannol provided protection against hydrogen peroxide induced DNA damage, but pterostilbene and resveratrol increased DNA damage following hydrogen peroxide treatment. None of the compounds showed any effect on DNA repair. Overall, this study indicates there is merit for further research into the bioactive roles, including antioxidant capacity, of all three compounds. Such research may provide evidence for the more widespread use of these and other food based compounds for preventing lifestyle diseases.

Bovine reproductive performance is one of the most important factors influencing dairy farm profitability. Present-day techniques for oestrus- and pregnancy-detection are unreliable and labour-intensive. Although measuring milk-progesterone at regular intervals allows the fertility status of a cow to be determined reliably, the labour cost of collecting and analysing samples is prohibitive. This project aimed to develop a progesterone sensing system that could be automated and integrated with the milking unit, thus minimising labour costs. The proposed system involved mixing the milk sample with an enzyme-antibody conjugate and then passing the sample through a column containing immobilised progesterone. Any progesterone in the milk would inhibit conjugate binding to the column. An enzyme substrate would then flow through the column and bound conjugate would be detected as a colour change at the column's outlet. Periodate-coupling was used to attach horseradish peroxidase enzyme to anti-progesterone antibody, and progesterone-3-carboxymethyloxime was immobilised on the polystyrene bead surface using amine-coupling. Both techniques are widely used. Initial experiments attempted to verify the success of these two reactions simultaneously, whereas later experiments focused on the bead-coating. Beads were suspended in a specially-constructed syringe and the antibody activity of the eluted solution was measured by SPR. However, a combination of non-specific binding and antibody stability and activity issues meant neither reaction was conclusively verified. Many trials were done to investigate how to overcome the problems encountered but a suitable, workable procedure was not developed. Despite poor progress, the problems encountered did not undermine the project's potential. There remains optimism of developing an on-line method if research were to continue.

This thesis reports an investigation from the “bedside” back to the “bench”. That is, from the economic evaluation of a medical intervention to basic research and development of a contractility assay. The underlying theme of this thesis is cellular contractility, which was stimulated from our laboratory’s work in the microvascular complications of Diabetic Retinopathy (DR). The health economic perspective of this thesis evaluates the cost effectiveness and cost utility of DR prognosis using the prog-DR test. This novel prognostic test developed in our laboratory relies on the contractile response of blood vessels to detect subjects with high risk of developing DR. Markov modeling based on information in the literature was used to estimate the outcomes of a hypothetical population. The costs, health and utility outcomes of DR were compared to the potential outcomes if the prog-DR test was used. The model show that the prog-DR test can improve the health of the hypothetical population as measured in the number of life years (LY), sight years (SY) and quality-adjusted life years (QALY). The prog-DR test was more cost effective than the benchmark of annual or bi-annual screening and the incremental cost effectiveness ratio (ICER) appears to be at an acceptable level. Scenario and sensitivity analysis also show that the cost effectiveness of the prog-DR test can be improved by (i) better blood glucose management post prog-DR test, (ii) targeted screening (as opposed to population-wide screening) and (iii) reduced costs of both screening and management of DM and DR. The physiological perspective of the thesis aimed to develop a contractility assay for DR that was based on a 3D scaffold, which was affordable, easy to make and mimicked the three dimensional physiological environment of blood vessels. The contractility assay was developed using a 3D, hollow scaffold (PE-PAH capsule) and involved (i) the selection of the optimal core material, (ii) optimisation of the manufacturing process, (iii) characterisation of the scaffold and (iv) ensuring that cells can be grown on it. The cyto-biocompatibility of the candidate polyelectrolyte Poly(Sodium 4-Styrene Sulfonate) (PSS) and Poly(Allylamine Hydrochloride) (PAH) in the thin films format were investigated using three different cell lines and the effects of these thin films were also compared to titanium and titanium nitride thin films. In essence, PSS and PAH are not cytotoxic and was used to develop the contractile scaffold, PE-PAH capsule. This scaffold is relative elastic and the contractile force exerted by the 3T3-L1 cells was calculated based on the deformation of the PE-PAH capsule. The contractility assay was sufficiently sensitive to detect the nano-Newton magnitude of force developed by individual cells and discriminated the change in force due to disruption of the F-actin cytoskeleton by forskolin and cytochalasin D.

Erlangen, Nürnberg, Universiẗat, Diss., 2007. / Enth. 1 Sonderabdr. aus: Journal of investigative dermatology ; 124. 2005. - Beitr. teilw. dt., teilw. engl.