Kinin-B2 Receptor Activity Determines the Differentiation Fate of Neural Stem Cells

Bradykinin is not only important for inflammation and blood pressure regulation, but also involved in neuromodulation and neuroprotection. Here we describe novel functions for bradykinin and the kinin-B2 receptor (B2BkR) in differentiation of neural stem cells. In the presence of the B2BkR antagonist HOE-140 during rat neurosphere differentiation, neuron-specific beta 3-tubulin and enolase expression was reduced together with an increase in glial protein expression, indicating that bradykinin- induced receptor activity contributes to neurogenesis. In agreement, HOE-140 affected in the same way expression levels of neural markers during neural differentiation of murine P19 and human iPS cells. Kinin-B1 receptor agonists and antagonists did not affect expression levels of neural markers, suggesting that bradykinin-mediated effects are exclusively mediated via B2BkR. Neurogenesis was augmented by bradykinin in the middle and late stages of the differentiation process. Chronic treatment with HOE-140 diminished eNOS and nNOS as well as M1-M4 muscarinic receptor expression and also affected purinergic receptor expression and activity. Neurogenesis, gliogenesis, and neural migration were altered during differentiation of neurospheres isolated from B2BkR knock-out mice. Whole mount in situ hybridization revealed the presence of B2BkR mRNA throughout the nervous system in mouse embryos, and less beta 3-tubulin and more glial proteins were expressed in developing and adult B2BkR knock-out mice brains. As a underlying transcriptional mechanism for neural fate determination, HOE-140 induced up-regulation of Notch1 and Stat3 gene expression. Because pharmacological treatments did not affect cell viability and proliferation, we conclude that bradykinin-induced signaling provides a switch for neural fate determination and specification of neurotransmitter receptor expression.

Evaluation of Sperm DNA Peroxidation in Fertile and Subfertile Dogs

Contents Oxidative stress (OS) has been recognized as one of the most important causes of male infertility. The antioxidant activities of seminal plasma and epididymal fluid are not enough to prevent OS, which can damage sperm membranes and DNA, so antioxidant supplementation has been used as a treatment of male infertility. The aim of this experiment was to evaluate the DNA peroxidation before and after antioxidant supplementation with vitamin C and E in dogs with and without fertility problems. A total of eleven dogs were used and were divided in two groups: fertile group (G1), dogs with normal spermiogram (n=5); subfertile group (G2): dogs with low sperm count (<20x106sptz/ml) and/or more than 30% of total sperm pathology (n=6). Both groups received 500mg/day of vitamin C and 500mg/day of vitamin E for 60days. A semen sample was collected before (M1) and after (M2) oral supplementation. Samples were analysed for DNA peroxidation by measuring the 8-hydroxy-2'-deoxyguanosine concentration. No significant difference was observed between groups at either time. Oral supplementation with 500mg/day of vitamin C and 500mg/day of vitamin E did not change the DNA peroxidation in fertile and subfertile dogs.

Profiling Three-Dimensional Nuclear Telomeric Architecture of Myelodysplastic Syndromes and Acute Myeloid Leukemia Defines Patient Subgroups

Purpose: Myelodysplastic syndromes (MDS) are a group of disorders characterized by cytopenias, with a propensity for evolution into acute myeloid leukemias (AML). This transformation is driven by genomic instability, but mechanisms remain unknown. Telomere dysfunction might generate genomic instability leading to cytopenias and disease progression. Experimental Design: We undertook a pilot study of 94 patients with MDS (56 patients) and AML (38 patients). The MDS cohort consisted of refractory cytopenia with multilineage dysplasia (32 cases), refractory anemia (12 cases), refractory anemia with excess of blasts (RAEB) 1 (8 cases), RAEB2 (1 case), refractory anemia with ring sideroblasts (2 cases), and MDS with isolated del(5q) (1 case). The AML cohort was composed of AML-M4 (12 cases), AML-M2 (10 cases), AML-M5 (5 cases), AML-M0 (5 cases), AML-M1 (2 cases), AML-M4eo (1 case), and AML with multidysplasia-related changes (1 case). Three-dimensional quantitative FISH of telomeres was carried out on nuclei from bone marrow samples and analyzed using TeloView. Results: We defined three-dimensional nuclear telomeric profiles on the basis of telomere numbers, telomeric aggregates, telomere signal intensities, nuclear volumes, and nuclear telomere distribution. Using these parameters, we blindly subdivided the MDS patients into nine subgroups and the AML patients into six subgroups. Each of the parameters showed significant differences between MDS and AML. Combining all parameters revealed significant differences between all subgroups. Three-dimensional telomeric profiles are linked to the evolution of telomere dysfunction, defining a model of progression from MDS to AML. Conclusions: Our results show distinct three-dimensional telomeric profiles specific to patients with MDS and AML that help subgroup patients based on the severity of telomere dysfunction highlighted in the profiles. Clin Cancer Res; 18(12); 3293-304. (C) 2012 AACR.

The nitrogen sufficiency index underlying estimates of nitrogen fertilization requirements of common bean

Chlorophyll determination with a portable chlorophyll meter can indicate the period of highest N demand of plants and whether sidedressing is required or not. In this sense, defining the optimal timing of N application to common bean is fundamental to increase N use efficiency, increase yields and reduce the cost of fertilization. The objectives of this study were to evaluate the efficiency of N sufficiency index (NSI) calculated based on the relative chlorophyll index (RCI) in leaves, measured with a portable chlorophyll meter, as an indicator of time of N sidedressing fertilization and to verify which NSI (90 and 95 %) value is the most appropriate to indicate the moment of N fertilization of common bean cultivar Perola. The experiment was carried out in the rainy and dry growing seasons of the agricultural year 2009/10 on a dystroferric Red Nitosol, in Botucatu, São Paulo State, Brazil. The experiment was arranged in a randomized complete block design with five treatments, consisting of N managements (M1: 200 kg ha-1 N (40 kg at sowing + 80 kg 15 days after emergence (DAE) + 80 kg 30 DAE); M2: 100 kg ha-1 N (20 kg at sowing + 40 kg 15 DAE + 40 kg 30 DAE); M3: 20 kg ha-1 N at sowing + 30 kg ha-1 when chlorophyll meter readings indicated NSI < 95 %; M4: 20 kg ha-1 N at sowing + 30 kg ha-1 N when chlorophyll meter readings indicated NSI < 90 % and, M5: control (without N application)) and four replications. The variables RCI, aboveground dry matter, total leaf N concentration, production components, grain yield, relative yield, and N use efficiency were evaluated. The RCI correlated with leaf N concentrations. By monitoring the RCI with the chlorophyll meter, the period of N sidedressing of common bean could be defined, improving N use efficiency and avoiding unnecessary N supply to common bean. The NSI 90 % of the reference area was more efficient to define the moment of N sidedressing of common bean, to increase N use efficiency.

X-Ray analysis to assess mechanical damage in sweet corn seeds

The X-ray test is a precise, fast and non-destructive method to detect mechanical damage in seeds. In the present study, the efficiency of X-ray analysis in identifying the extent of mechanical damage in sweet corn seeds and its relationship with germination and vigor was evaluated. Hybrid 'SWB 551' (sh2) seeds with round (R) and flat (F) shapes were classified as large (L), medium (M1, M2 and M3) and small (S), using sieves with round and oblong screens. After artificial exposure to different levels of damage (0, 1, 3, 5 and 7 impacts), seeds were X-rayed (15 kV, 5 min) and submitted to germination (25 °C/5 days) and cold (10 °C/7 days) tests. Digital images of normal and abnormal seedlings and ungerminated seeds from germination and cold tests were jointly analyzed with the seed X-ray images. Results showed that damage affecting the embryonic axis resulted in abnormal seedlings or dead seeds in the germination and cold tests. The X-ray analysis is efficient for identifying mechanical damage in sweet corn seeds, allowing damage severity to be associated with losses in germination and vigor.

Relative CO(2)/NH(3) selectivities of mammalian aquaporins 0-9

Previous work showed that aquaporin 1 (AQP1), AQP4-M23, and AQP5 each has a characteristic CO(2)/NH(3) and CO(2)/H(2)O permeability ratio. The goal of the present study is to characterize AQPs 0-9, which traffic to the plasma membrane when heterologously expressed in Xenopus oocytes. We use video microscopy to compute osmotic water permeability (P(f)) and microelectrodes to record transient changes in surface pH (ΔpH(S)) caused by CO(2) or NH(3) influx. Subtracting respective values for day-matched, H(2)O-injected control oocytes yields the channel-specific values P(f)* and ΔpH(S)*. We find that P(f)* is significantly >0 for all AQPs tested except AQP6. (ΔpH(S)*)(CO(2)) is significantly >0 for AQP0, AQP1, AQP4-M23, AQP5, AQP6, and AQP9. (ΔpH(S)*)(NH(3)) is >0 for AQP1, AQP3, AQP6, AQP7, AQP8, and AQP9. The ratio (ΔpH(S)*)(CO(2))/P(f)* falls in the sequence AQP6 (∞) > AQP5 > AQP4-M23 > AQP0 ≅ AQP1 ≅ AQP9 > others (0). The ratio (ΔpH(S)*)(NH(3))/P(f)* falls in the sequence AQP6 (∞) > AQP3 ≅ AQP7 ≅ AQP8 ≅ AQP9 > AQP1 > others (0). Finally, the ratio (ΔpH(S)*)(CO(2))/(-ΔpH(S)*)(NH(3)) falls in the sequence AQP0 (∞) ≅ AQP4-M23 ≅ AQP5 > AQP6 > AQP1 > AQP9 > AQP3 (0) ≅ AQP7 ≅ AQP8. The ratio (ΔpH(S)*)(CO(2))/(-ΔpH(S)*)(NH(3)) is indeterminate for both AQP2 and AQP4-M1. In summary, we find that mammalian AQPs exhibit a diverse range of selectivities for CO(2) vs. NH(3) vs. H(2)O. As a consequence, by expressing specific combinations of AQPs, cells could exert considerable control over the movements of each of these three substances

Image analysis in the morphological and morphometric study of teeth

The subject of this doctoral dissertation concerns the definition of a new methodology for the morphological and morphometric study of fossilized human teeth, and therefore strives to provide a contribution to the reconstruction of human evolutionary history that proposes to extend to the different species of hominid fossils. Standardized investigative methodologies are lacking both regarding the orientation of teeth subject to study and in the analysis that can be carried out on these teeth once they are oriented. The opportunity to standardize a primary analysis methodology is furnished by the study of certain early Neanderthal and preneanderthal molars recovered in two caves in southern Italy [Grotta Taddeo (Taddeo Cave) and Grotta del Poggio (Poggio Cave), near Marina di Camerata, Campania]. To these we can add other molars of Neanderthal and modern man of the upper Paleolithic era, specifically scanned in the paleoanthropology laboratory of the University of Arkansas (Fayetteville, Arkansas, USA), in order to increase the paleoanthropological sample data and thereby make the final results of the analyses more significant. The new analysis methodology is rendered as follows: 1. Standardization of an orientation system for primary molars (superior and inferior), starting from a scan of a sample of 30 molars belonging to modern man (15 M1 inferior and 15 M1 superior), the definition of landmarks, the comparison of various systems and the choice of a system of orientation for each of the two dental typologies. 2. The definition of an analysis procedure that considers only the first 4 millimeters of the dental crown starting from the collar: 5 sections parallel to the plane according to which the tooth has been oriented are carried out, spaced 1 millimeter between them. The intention is to determine a method that allows for the differentiation of fossilized species even in the presence of worn teeth. 3. Results and Conclusions. The new approach to the study of teeth provides a considerable quantity of information that can better be evaluated by increasing the fossil sample data. It has been demonstrated to be a valid tool in evolutionary classification that has allowed (us) to differentiate the Neanderthal sample from that of modern man. In a particular sense the molars of Grotta Taddeo, which up until this point it has not been possible to determine with exactness their species of origin, through the present research they are classified as Neanderthal.

Mechanismen der Aktivierung und Detoxifizierung von Aflatoxin B 1 in verschiedenen Leberzelltypen von Ratten, Mäusen und Waldmurmeltieren

Zusammenfassung: Die Applikation des Mykotoxins Aflatoxin B1 (AFB1) führt in der Ratte zu Lebertumoren hepatozellulären Ursprungs, während bisher keine transformierende Wirkung dieses Mykotoxins auf Kupffer- und Endothelzellen (Nichtparenchymzellen, NPC) nachgewiesen werden konnte. Diese Resistenzmechanismen der NPC gegenüber AFB1 wurden im ersten Teil dieser Arbeit untersucht. AFB1 ist per se inaktiv, wird jedoch durch Verstoffwechselung in den chemisch reaktiven, an DNA bindenden Metaboliten AFB1-8,9-Epoxid überführt. Daneben stellt die enzymatische Hydroxylierung von AFB1 am Kohlenstoff-9a zum Aflatoxin M1 eine Detoxifizierung dar. Durch HPLC-Analyse der AFB1-Metabolite konnte gezeigt werden, daß in Nichtparenchymzellen (NPC) das Verhältnis von 9a-Hydroxylierung zu 8,9-Epoxidierung höher als in Parenchymzellen (PC) ist. Die AFB1-9a-hydroxylase fördert insbesondere in den NPC der Leber die Bildung des weniger gentoxischen Metaboliten AFM1 und konkurriert daher um die Aktivierung von AFB1 zum mutagenen und kanzerogenen 8,9-Epoxid. Dieser metabolische Unterschied scheint also einen Beitrag zur Resistenz der NPC der Leber gegenüber der hepatokanzerogenen Wirkung von AFB1 zu leisten. Da ein Synergismus zwischen der AFB1-Exposition und einer Infektion mit dem Hepatitis B-Virus (HBV) beim Menschen bezüglich des Auftretens von hepatozellulären Karzinomen zu bestehen scheint, wurde im zweiten Teil dieser Arbeit untersucht, ob die metabolische Aktivierung von AFB1 durch eine HBV-Infektion verstärkt wird. In einem Vergleich der Biotransformation von AFB1 mit mikrosomalen Leberfraktionen von transgenen HBV-Mäusen und Kontrollmäusen wurde keine signifikanten Unterschiede festgestellt. Dagegen wurde bei Virus-infizierten Waldmurmeltieren eine deutlich reduzierte Bildung des AFB1-8,9-Epoxids beobachtet. Es konnte z.T. ein Zusammenhang zwischen den verschiedenen Stadien der Leberschädigung und den Metabolismusraten festgestellt werden, wobei die metabolische Aktivierung mit zunehmender Leberschädigung abzunehmen scheint. Auch hinsichtlich der Aktivitäten verschiedener Cytochrom P450 abhängiger Monooxygenasen wurde eine weitgehende Übereinstimmung mit den durch HPLC ermittelten Metabolitenprofilen des AFB1 beobachtet. Diese Studien mit subzellulären Leberfraktion der transgenen HBV-Mäusen und der Waldmurmeltieren zeigen, daß die Interaktion zwischen Hepatitis und AFB1 nicht mit der verstärkten metabolischer Aktivierung von AFB1 zu erklären ist. TGF-ß1, aus der Gruppe der Cytokine, wird als Mediator bei Entzündungsprozessen in der Leber so z.B. im Verlauf einer Virushepatitis freigesetzt. Aufgrund der besonderen Bedeutung des murinen CYP2A5 (ortholog zum humanen CYP2A6) bei der Aktivierung von AFB1 wurde der Einfluß von TGF-ß1 auf CYP2A5 in Primärkulturen von Maushepatozyten untersucht. Durch Messung der Aktivität der Cumarin-7-hydroxylase sowie durch Bestimmung der Proteinmenge von CYP2A5 mittels Western Blotting konnte zunächst die Induzierbarkeit des CYP2A5-Isoenzyms durch Phenobarbital in kultivierten Hepatozyten der Maus gezeigt werden. Nur bei einer niedrigen TGF-ß1-Konzentration wurde eine leicht erhöhte Expression von CYP2A5 festgestellt, ansonsten führte die Behandlung der kultivierten Maushepatozyten mit TGF-ß1 zu einer dosisabhängigen Verminderung der Expression von CYP2A5.

Laserspektroskopie der 1s-Hyperfeinstrukturaufspaltung an wasserstoffähnlichem 207 Pb 81+

Die Messung der 1s Hyperfeinstruktur (HFS) wasserstoffähn-licher Ionen bei hoher Kernladung Z erlaubt den Test der QED in Gegenwart starker elektrischer und magnetischer Felder durchzuführen. Aufgrund der Z^3-Abhängigkeit der magnetischen Wechselwirkung liegt die 1s-HFS bei hohem Z im optischen Spektrum und kann mit der Laserspektroskopie untersucht werden.In der vorliegenden Arbeit wurde die Grundzustands-HFS an 207Pb81+ bestimmt. Die Experimente wurden am Speicherring ESR der Gesellschaft für Schwerionenforschung mbH am Elek-tronen-gekühlten Bleistrahl durchgeführt.Ein besonderer Schwerpunkt dieser Arbeit war die Entwick-lung neuer Experimentiertechniken, die es erlauben, lang-lebigen Strahlungsübergänge im nahen infraroten Spektral-bereich mit Hilfe der Fluoreszenz-Laserspektroskopie am Speicherring zu untersuchen. Der Ionenstrahl wurde in kollinearer Geometrie mit einem Nd:YAG-Laser angeregt und die Resonanz durch Doppler-Abstimmung gemessen. Der M1-Übergang der 1s HFS an 207Pb81+ liegt bei 1019,7(2) nm. Die QED-Korrekturen sind damit auf wenige Prozent genau bestimmt. Die theoretische Berechnung der QED-Korrekturen benutzt die gemessenen bzw. den Kernmodellen entnommenen Verteilungen der Kernladung und der Kernmagnetisierung. Die Unsicherheit der Kenntnis dieser Verteilungen spielt nur eine untergeordnete Rolle für die Fehler in der gerechneten QED-Korrektur. Allerdings existieren für das magnetische Moment von 207Pb zwei widersprüchliche Literaturwerte. Die Diskussion der Ergebnisse wird im Rahmen dieses Sachver-haltes geführt.Die natürliche Lebensdauer des oberen Hyperfein-Niveaus wurde mit 49,5(6,5) ms gemessen und ist mit dem Theoriewert von 52,3(2) ms verträglich. Durch präzise Lebensdauermes-sungen können QED-Korrekturen zum g-Faktor des gebundenen Elektrons getestet werden.

Photoproduktion neutraler Pionen am Proton mit linear polarisierten Photonen im Bereich der Delta(1232)-Resonanz

Hinweise auf die innere Struktur des Nukleons, dessenbeobachtbares Quadrupolmoment verschwindet, lassen sich u.a.durch die Untersuchung des N->Delta(1232)-Übergangsgewinnen. Dieser wird von der magnetischen Dipolanregung M1- einem Spinflip-Übergang - dominiert. In der Reaktion (gamma(lin. pol.) p -> p pi0) gelingt es mittels der Photonasymmetrie Sigma, das Signal der kleinen elektrischen Quadrupolamplitude E2 in einem Interferenztermmit der M1-Amplitude zu verstärken und das VerhältnisREM=E2/M1 des betrachteten Übergangs zu bestimmen. DieE2-Amplitude des N->Delta-Übergangs läßt auf eineDeformation des Nukleons und/oder der Delta-Resonanzschließen. Das zugehörige Experiment wurde am MainzerElektronenbeschleuniger MAMI durchgeführt. Durch kohärenteBremsstrahlung der Elektronen an einem Diamantradiatorstanden im Bereich der Delta(1232)-Resonanz linear polarisierte Photonen zur Verfügung. Insgesamt wurden reellePhotonen im Bereich Egamma=(200-790) MeV von derA2-Photonenmarkierungsanlage (Glasgow--Tagger)energiemarkiert. Mit dem Photonenspektrometer TAPS wurden die pi0-Mesonen über ihre beiden Zerfallsphotonennachgewiesen. Die gewählte Anordnung der 504BaF2-Einzelkristalle um ein Flüssigwasserstofftargeterlaubte den pi0-Nachweis im vollen Polarwinkelbereich. Die Datenbasis zur pi0-Photoproduktion am Proton konntehinsichtlich der Wirkungsquerschnitte und Photonasymmetriendurch Datenpunkte über den gesamten Polarwinkelbereichhinweg nachdrücklich erweitert werden.Eine weiterführende Multipolanalyse der neuen(Proton-pi0)-Daten ermöglichte im Energiebereich derDelta-Resonanz die Bestimmung der s- und p-WellenIsospinamplituden von E0+, M1-, E1+ und M1+.

Previsione del comportamento dinamico e sismico di un edificio a tre piani in scala reale costituito da pareti sandwich in C.A. gettato in opera da provare su tavola vibrante

INDICE INTRODUZIONE 1 1. DESCRIZIONE DEL SISTEMA COSTRUTTIVO 5 1.1 I pannelli modulari 5 1.2 Le pareti tozze in cemento armato gettate in opera realizzate con la tecnologia del pannello di supporto in polistirene 5 1.3 La connessione tra le pareti e la fondazione 6 1.4 Le connessioni tra pareti ortogonali 7 1.5 Le connessioni tra pareti e solai 7 1.6 Il sistema strutturale così ottenuto e le sue caratteristiche salienti 8 2. RICERCA BIBLIOGRAFICA 11 2.1 Pareti tozze e pareti snelle 11 2.2 Il comportamento scatolare 13 2.3 I muri sandwich 14 2.4 Il “ferro-cemento” 15 3. DATI DI PARTENZA 19 3.1 Schema geometrico - architettonico definitivo 19 3.2 Abaco delle sezioni e delle armature 21 3.3 Materiali e resistenze 22 3.4 Valutazione del momento di inerzia delle pareti estese debolmente armate 23 3.4.1 Generalità 23 3.4.2 Caratteristiche degli elementi provati 23 3.4.3 Formulazioni analitiche 23 3.4.4 Considerazioni sulla deformabilità dei pannelli debolmente armati 24 3.4.5 Confronto tra rigidezze sperimentali e rigidezze valutate analiticamente 26 3.4.6 Stima di un modulo elastico equivalente 26 4. ANALISI DEI CARICHI 29 4.1 Stima dei carichi di progetto della struttura 29 4.1.1 Stima dei pesi di piano 30 4.1.2 Tabella riassuntiva dei pesi di piano 31 4.2 Analisi dei carichi da applicare in fase di prova 32 4.2.1 Pesi di piano 34 4.2.2 Tabella riassuntiva dei pesi di piano 35 4.3 Pesi della struttura 36 4.3.1 Ripartizione del carico sulle pareti parallele e ortogonali 36 5. DESCRIZIONE DEL MODELLO AGLI ELEMENTI FINITI 37 5.1 Caratteristiche di modellazione 37 5.2 Caratteristiche geometriche del modello 38 5.3 Analisi dei carichi 41 5.4 Modello con shell costituite da un solo layer 43 5.4.1 Modellazione dei solai 43 5.4.2 Modellazione delle pareti 44 5.4.3 Descrizione delle caratteristiche dei materiali 46 5.4.3.1 Comportamento lineare dei materiali 46 6. ANALISI DEL COMPORTAMENTO STATICO DELLA STRUTTURA 49 6.1 Azioni statiche 49 6.2 Analisi statica 49 7. ANALISI DEL COMPORTAMENTO DINAMICO DELLA STRUTTURA 51 7.1 Determinazione del periodo proprio della struttura con il modello FEM 51 7.1.1 Modi di vibrare corrispondenti al modello con solai e pareti costituiti da elementi shell 51 7.1.1.1 Modi di vibrare con modulo pari a E 51 7.1.1.2 Modi di vibrare con modulo pari a 0,5E 51 7.1.1.3 Modi di vibrare con modulo pari a 0,1E 51 7.1.2 Modi di vibrare corrispondenti al modello con solai infinitamente rigidi e pareti costituite da elementi shell 52 7.1.2.1 Modi di vibrare con modulo pari a E 52 7.1.2.2 Modi di vibrare con modulo pari a 0,5E 52 7.1.2.3 Modi di vibrare con modulo pari a 0,1E: 52 7.1.3 Modi di vibrare corrispondenti al modello con solai irrigiditi con bielle e pareti costituite da elementi shell 53 7.1.3.1 Modi di vibrare con modulo pari a E 53 7.1.3.2 Modi di vibrare con modulo pari a 0,5E 53 7.1.3.3 Modi di vibrare con modulo pari a 0,1E 53 7.2 Calcolo del periodo proprio della struttura assimilandola ad un oscillatore semplice 59 7.2.1 Analisi svolta assumendo l’azione del sisma in ingresso in direzione X-X 59 7.2.1.1 Analisi svolta assumendo il modulo elastico E pari a 300000 Kg/cm2 59 7.2.1.1.1 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H e modulo elastico assunto pari ad E 59 7.2.1.1.2 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H e modulo elastico assunto pari ad E 61 7.2.1.1.3 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H, modulo elastico assunto pari ad E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 63 7.2.1.1.4 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H, modulo elastico assunto pari ad E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 66 7.2.1.2 Analisi svolta assumendo il modulo elastico E pari a 150000 Kg/cm2 69 7.2.1.2.1 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H e modulo elastico assunto pari a 0,5E 69 7.2.1.2.2 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H e modulo elastico assunto pari a 0,5E 71 7.2.1.2.3 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H, modulo elastico assunto pari a 0,5 E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 73 7.2.1.2.4 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H, modulo elastico assunto pari a 0,5 E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 76 7.2.1.3 Analisi svolta assumendo il modulo elastico E pari a 30000 Kg/cm2 79 7.2.1.3.1 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H e modulo elastico assunto pari a 0,1E 79 7.2.1.3.2 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H e modulo elastico assunto pari a 0,1E 81 7.2.1.3.3 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H, modulo elastico assunto pari a 0,1E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 83 7.2.1.3.4 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H, modulo elastico assunto pari a 0,1E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 86 7.2.2 Analisi svolta assumendo l’azione del sisma in ingresso in direzione Y-Y 89 7.2.2.1 Analisi svolta assumendo il modulo elastico E pari a 300000 Kg/cm2 89 7.2.2.1.1 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H e modulo elastico assunto pari ad E 89 7.2.2.1.2 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H e modulo elastico assunto pari ad E 91 7.2.2.1.3 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H, modulo elastico assunto pari ad E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 93 7.2.2.1.4 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H, modulo elastico assunto pari ad E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 98 7.2.2.1.5 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H e modulo elastico assunto pari ad E 103 7.2.2.1.6 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H e modulo elastico assunto pari ad E 105 7.2.2.1.7 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H, modulo elastico assunto pari ad E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 107 7.2.2.1.8 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H, modulo elastico assunto pari ad E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 112 7.2.2.2 Analisi svolta assumendo il modulo elastico E pari a 150000 Kg/cm2 117 7.2.2.2.1 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H e modulo elastico assunto pari a 0,5E 117 7.2.2.2.2 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H e modulo elastico assunto pari a 0,5E 119 7.2.2.2.3 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H, modulo elastico assunto pari a 0,5 E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 121 7.2.2.2.4 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H, modulo elastico assunto pari a 0,5 E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 126 7.2.2.2.5 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H e modulo elastico assunto pari a 0,5 E 131 7.2.2.2.6 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H e modulo elastico assunto pari ad E 133 7.2.2.2.7 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H, modulo elastico assunto pari a 0,5E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 135 7.2.2.2.8 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H, modulo elastico assunto pari a 0,5E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 140 7.2.2.3 Analisi svolta assumendo il modulo elastico E pari a 30000 Kg/cm2 145 7.2.2.3.1 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H e modulo elastico assunto pari a 0,1E 145 7.2.2.3.2 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H e modulo elastico assunto pari a 0,1E 147 7.2.2.3.3 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H, modulo elastico assunto pari a 0,1E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 149 7.2.2.3.4 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H, modulo elastico assunto pari a 0,1E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 154 7.2.2.3.5 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H e modulo elastico assunto pari a 0,1 E 159 7.2.2.3.6 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H e modulo elastico assunto pari ad E 161 7.2.2.3.7 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 2/3 H, modulo elastico assunto pari a 0,1E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 163 7.2.2.3.8 Determinazione del periodo proprio della struttura considerando la massa complessiva concentrata a 1/2 H, modulo elastico assunto pari a 0,1E, e struttura resistente costituita dai soli “maschi murari” delle pareti parallele all’azione del sisma 168 7.3 Calcolo del periodo proprio della struttura approssimato utilizzando espressioni analitiche 174 7.3.1 Approssimazione della struttura ad una mensola incastrata di peso Q=ql avente un peso P gravante all’estremo libero 174 7.3.1.1 Riferimenti teorici: sostituzione di masse distribuite con masse concentrate 174 7.3.1.2 Applicazione allo specifico caso di studio in esame con modulo elastico E=300000 kg/cm2 177 7.3.1.3 Applicazione allo specifico caso di studio in esame con modulo elastico E=30000 kg/cm2 179 7.3.2 Approssimazione della struttura ad una mensola incastrata alla base, di peso Q=ql, avente un peso P gravante all’estremo libero e struttura resistente costituita dai soli “maschi murari”delle pareti parallele all’azione del sisma 181 7.3.2.1 Applicazione allo specifico caso di studio in esame con modulo elastico E=300000 kg/cm2 181 7.3.2.2 Applicazione allo specifico caso di studio in esame con modulo elastico E=30000 kg/cm2 186 7.3.3 Approssimazione della struttura ad un portale avente peso Qp = peso di un piedritto, Qt=peso del traverso e un peso P gravante sul traverso medesimo 191 7.3.3.1 Riferimenti teorici: sostituzione di masse distribuite con masse concentrate 191 7.3.3.2 Applicazione allo specifico caso di studio in esame con modulo ellastico E=300000 kg/cm2 192 7.3.3.3 Applicazione allo specifico caso di studio in esame con modulo ellastico E=30000 kg/cm2 194 7.3.4 Approssimazione della struttura ad un portale di peso Qp = peso di un piedritto, Qt=peso del traverso e avente un peso P gravante sul traverso medesimo e struttura resistente costituita dai soli “maschi murari”delle pareti parallele all’azione del sisma 196 7.3.4.1 Applicazione allo specifico caso di studio in esame con modulo elastico E=300000 kg/cm2 196 7.3.4.2 Applicazione allo specifico caso di studio in esame con modulo elastico E=30000 kg/cm2 201 7.3.5 Approssimazione della struttura ad una mensola incastrata di peso Q=ql avente le masse m1,m2....mn concentrate nei punti 1,2….n 206 7.3.5.1 Riferimenti teorici: metodo approssimato 206 7.3.5.2 Applicazione allo specifico caso di studio in esame con modulo elastico E=300000 kg/cm2 207 7.3.5.3 Applicazione allo specifico caso di studio in esame con modulo elastico E=30000 kg/cm2 209 7.3.6 Approssimazione della struttura ad un telaio deformabile con tavi infinitamente rigide 211 7.3.6.1 Riferimenti teorici: vibrazioni dei telai 211 7.3.6.2 Applicazione allo specifico caso di studio in esame con modulo elastico E=300000 kg/cm2 212 7.3.6.3 Applicazione allo specifico caso di studio in esame con modulo elastico E=30000 kg/cm2 215 7.3.7 Approssimazione della struttura ad una mensola incastrata di peso Q=ql avente masse m1,m2....mn concentrate nei punti 1,2….n e studiata come un sistema continuo 218 7.3.7.1 Riferimenti teorici: metodo energetico; Masse ripartite e concentrate; Formula di Dunkerley 218 7.3.7.1.1 Il metodo energetico 218 7.3.7.1.2 Masse ripartite e concentrate. Formula di Dunkerley 219 7.3.7.2 Applicazione allo specifico caso di studio in esame con modulo elastico E=300000 kg/cm2 221 7.3.7.3 Applicazione allo specifico caso di studio in esame con modulo elastico E=30000 kg/cm2 226 7.4 Calcolo del periodo della struttura approssimato mediante telaio equivalente 232 7.4.1 Dati geometrici relativi al telaio equivalente e determinazione dei carichi agenti su di esso 232 7.4.1.1 Determinazione del periodo proprio della struttura assumendo diversi valori del modulo elastico E 233 7.5 Conclusioni 234 7.5.1 Comparazione dei risultati relativi alla schematizzazione dell’edificio con una struttura ad un grado di libertà 234 7.5.2 Comparazione dei risultati relativi alla schematizzazione dell’edificio con una struttura a più gradi di libertà e a sistema continuo 236 8. ANALISI DEL COMPORTAMENTO SISMICO DELLA STRUTTURA 239 8.1 Modello con shell costituite da un solo layer 239 8.1.1 Analisi dinamica modale con spettro di risposta avente un valore di PGA pari a 0,1g 239 8.1.1.1 Generalità 239 8.1.1.2 Sollecitazioni e tensioni sulla sezione di base 242 8.1.1.2.1 Combinazione di carico ”Carichi verticali più Spettro di Risposta scalato ad un valore di PGA pari a 0,1g” 242 8.1.1.2.2 Combinazione di carico ”Spettro di Risposta scalato ad un valore di 0,1g di PGA” 245 8.1.1.3 Spostamenti di piano 248 8.1.1.4 Accelerazioni di piano 248 8.1.2 Analisi Time-History lineare con accelerogramma caratterizzato da un valore di PGA pari a 0,1g 249 8.1.2.1 Generalità 249 8.1.2.2 Sollecitazioni e tensioni sulla sezione di base 251 8.1.2.2.1 Combinazione di carico ” Carichi verticali più Accelerogramma agente in direzione Ye avente una PGA pari a 0,1g” 251 8.1.2.2.2 Combinazione di carico ” Accelerogramma agente in direzione Y avente un valore di PGA pari a 0,1g ” 254 8.1.2.3 Spostamenti di piano assoluti 257 8.1.2.4 Spostamenti di piano relativi 260 8.1.2.5 Accelerazioni di piano assolute 262 8.1.3 Analisi dinamica modale con spettro di risposta avente un valore di PGA pari a 0,3g 264 8.1.3.1 Generalità 264 8.1.3.2 Sollecitazioni e tensioni sulla sezione di base 265 8.1.

Tetracycline-inducible RNAi Knockdown of SCL (Stem Cell Leukaemia) in Mice

RNAi (RNA interference) is a powerful technology for sequence-specific targeting of mRNAs. This thesis was aimed at establishing conditions for conditional RNAi-mediated silencing first in vitro and subsequently also in transgenic mice. As a target the basic helix-loop-helix transcription factor encoding gene SCL (stem cell leukaemia also known as Tal-1 or TCL5) was used. SCL is a key regulator for haematopoietic development and ectopic expression of SCL is correlated with acute T-lymphoblastic leukaemias. Loss of SCL function studies demonstrated that ab initio deletion of SCL resulted in embryonic lethality around day E9 in gestation. To be able to conditionally inactivate SCL, RNAi technology was combined with the tetracycline-dependent regulatory system. This strategy allowed to exogenously control the induction of RNAi in a reversible fashion and consequently the generation of a completely switchable RNAi knockdown. First a suitable vector allowing for co-expression of tetracycline-controlled shRNAs (small hairpin RNAs) and constitutively active EGFP (enhanced green fluorescent protein) was generated. This novel vector, pRNAi-EGFP, was then evaluated for EGFP expression and tetracycline-mediated expression of shRNAs. Four sequences targeting different regions within the SCL mRNA were tested for their efficiency to specifically knockdown SCL. These experiments were performed in M1 murine leukaemia cells and subsequently in the HEK 293 cell line, expressing an engineered HA-tagged SCL protein. The second assay provided a solid experimental method for determining the efficiency of different SCL-siRNA knockdown constructs in tissue culture. Western blotting analyses revealed a down regulation of SCL protein for all four tested SCL-specific target sequences albeit with different knockdown efficiencies (between 25% and 100%). Furthermore, stringent tetracycline-dependent switchability of shRNA expression was confirmed by co-transfecting the SCL-specific pRNAi-EGFP vector (SCL-siRNA) together with the HA-tagged SCL expression plasmid into the HEK 293TR /T-REx cell line constitutively expressing the tetracycline repressor (TetR). These series of experiments demonstrated tight regulation of siRNA expression without background activity. To be able to control the SCL knockdown in vivo and especially to circumvent any possible embryonic lethality a transgenic mouse line with general expression of a tetracycline repressor was needed. Two alternative methods were used to generate TetR mice. The first approach was to co-inject the tetracycline-regulated RNAi vector together with a commercially available and here specifically modified T-REx expression vector (SCL-siRNA T-REx FRT LoxP mouse line). The second method involved the generation of a TetR expressor mouse line, which was then used for donating TetR-positive oocytes for pronuclear injection of the RNAi vector (SCL-siRNA T-REx mouse line). As expected, and in agreement with data from conditional Cre-controlled adult SCL knockout mice, post-transcriptional silencing of SCL by RNAi caused a shift in the maturation of red blood cell populations. This was shown in the bone marrow and peripheral blood by FACS analysis with the red blood cell-specific TER119 and CD71 markers which can be used to define erythrocyte differentiation (Lodish plot technique). In conclusion this study established conditions for effective SCL RNAi-mediated silencing in vitro and in vivo providing an important tool for further investigations into the role of SCL and, more generally, of its in vivo function in haematopoiesis and leukaemia. Most importantly, the here acquired knowledge will now allow the establishment of other completely conditional and reversible knockdown phenotypes in mice.

Eine neue Form der Pyruvatkinase in Säugerspermien

Nach Homogenisation ejakulierter Eberspermien und Zentrifugation des Homogenates blieben mehr als 60% der Aktivität des glykolytischen Enzyms Pyruvatkinase (PK) an Zellfragmenten im Sediment gebunden. Diese strukturgebundene PK wurde als PK-S bezeichnet. Das Detergenz Triton X-100 führte nicht zur Ablösung der PK-S; mit Trypsin konnten jedoch rund 80% der PK-S ohne Verlust an Aktivität von den Strukturen gelöst und durch kombinierte Kationenaustausch- und Hydrophobizitätschromatographie gereinigt werden (spezifische Aktivität: 116,7 U/mg Protein). Die lösliche PK aus Eberspermien konnte ebenfalls durch ein ähnliches Verfahren angereichert werden. Im Gel (SDS-PAGE) zeigten die Untereinheiten der PK-S mit 64.400 eine geringfügig größere relative Molekülmasse als die der PK-M1 aus Kaninchenmuskel (62.000). Die kinetischen Eigenschaften der abgelösten PK-S als auch der noch an Spermienstrukturen gebundenen PK-S und der löslichen PK aus Eberspermien waren sehr ähnlich und entsprachen der M1-Isoform der PK. Antikörper gegen Kaninchenmuskel-PK (Anti-PK-M1) reagierten auch mit der löslichen PK und der PK-S aus Eberspermien. Edman-Abbau der ersten 19 Aminosäuren zeigte, dass die tryptisch abgelöste PK-S am N-Terminus um 5 Aminosäuren gegenüber nativer PK-M1 verlängert ist, während der C-Terminus der erhaltenen PK-S-Sequenz mit einem meist nahe dem N-Terminus gelegenen Sequenzabschnitt der PK-M1 und -M2 übereinstimmt. Die N-terminale Verlängerung der nativen PK-S enthält sicherlich mehr als die nach tryptischer Lyse nachgewiesenen 5 Aminosäuren. Vergleiche der Aminosäure- und übersetzten Nukleotidsequenzen sowie die kinetischen Eigenschaften lassen vermuten, dass die PK-S, wie die PK-M1 und PK-M2, vom PKM-Gen codiert wird. Gegen die gereinigte PK-S wurden Antikörper in Kaninchen produziert. Da das Antiserum nicht ausreichend spezifisch für PK-S war, wurden aus ihm affinitätschromatographisch Antikörper (Anti-PK-S) isoliert, die hohe Affinität zu einem synthetisierten PK-S-Peptid (13 N-terminale Aminosäuren der tryptisch abgelösten PK-S) hatten. Dieses Anti-PK-S-Präparat war spezifisch für PK-S; es reagierte weder mit Kaninchenmuskel-PK noch mit löslicher PK oder anderen Proteinen aus Eberspermien. Anti-PK-S und Anti-PK-M1 wurden zur Lokalisierung von PK-S und löslicher PK in Spermien von Eber, Bulle und Mensch sowie in Schnitten von Eberhoden eingesetzt. Mit Anti-PK-S wurden der Bereich des Akrosoms und das lange flagellare Hauptstück sowie der Übergangsbereich zwischen Kopf und Mittelstück von Eberspermien fluoreszenzmarkiert, wogegen das kurze, die Mitochondrien enthaltende Mittelstück des Flagellums und der postakrosomale Kopfbereich nur mit Anti-PK-M1 markiert wurden. Immunogoldmarkierung in elektronenmikroskopischen Bildern bestätigte die Lokalisierung von PK-S im Akrosombereich. Im Hauptstück banden Anti-PK-M1 und Anti-PK-S an die fibröse Scheide. Glyzerinaldehyd-3-phosphat Dehydrogenase (GAPDH) konnte von mir ebenfalls im Akrosombereich, im Übergangsbereich zwischen Kopf und Mittelstück und an der fibrösen Scheide detektiert werden. Auch an Bullen- und Humanspermien konnte über Immunogoldmarkierung PK und vermutlich GAPDH an der fibrösen Scheide gezeigt werden. Im Akrosombereich dieser Spermien waren die Nachweise von PK und GAPDH jedoch nicht sicher. In Eberhodenschnitten war die PK-S erstmals, oder zumindest vermehrt, in den elongierenden Spermatiden über Fluoreszenzmarkierung nachweisbar, während andere, vermutlich somatische PK vermehrt in den früheren Stadien (Spermatogonien, aber auch in den Spermatozyten und runden Spermatiden) auftrat. Für die GAPDH zeigte sich ein ähnlicher Entwicklungsverlauf. Die Ergebnisse zeigen, dass in Eberspermien zwei Isoformen der PK auftreten: eine N-terminal verlängerte, strukturgebundene Form, die PK-S, und eine lösliche Form, die beide der PK-M1 ähneln. Der ungewöhnliche N-Terminus der PK-S dient vermutlich der spezifischen räumlichen Anordnung der PK-S im Akrosombereich und an der fibrösen Scheide, nicht aber der Modulation kinetischer Eigenschaften. Meine Untersuchungen stützen die Hypothese, dass in bestimmten Kompartimenten von Säugerspermien die Glykolyse durch Verankerung einiger ihrer Enzyme strukturell hochgeordnet ist. Dadurch wird vermutlich die Versorgung der Mitochondrien-freien Regionen mit ATP sichergestellt. Man kann diese Organisation als Anpassung des Stoffwechsels von Spermien deuten, bei denen die Mitochondrien in einem kleinen Bereich (Mittelstück) hinter dem Spermienkopf kompartimentiert sind. Im Hauptstück des Flagellums könnte die Glykolyse ATP für die Spermienmotilität liefern, im Akrosombereich für die Verhinderung einer vorzeitigen Akrosomreaktion. Somit käme der strukturierten Glykolyse eine essentielle Bedeutung für die Befruchtungsfähigkeit von Säugerspermien zu.

Design and synthesis of multipotent drugs: application to Alzheimer's disease and benign prostatic hyperplasia

The aim of this thesis was to synthesize multipotent drugs for the treatment of Alzheimer’s disease (AD) and for benign prostatic hyperplasia (BPH), two diseases that affect the elderly. AD is a neurodegenerative disorder that is characterized, among other factors, by loss of cholinergic neurons. Selective activation of M1 receptors through an allosteric site could restore the cholinergic hypofunction, improving the cognition in AD patients. We describe here the discovery and SAR of a novel series of quinone derivatives. Among them, 1 was the most interesting, being a high M1 selective positive allosteric modulator. At 100 nM, 1 triplicated the production of cAMP induced by oxotremorine. Moreover, it inhibited AChE and it displayed antioxidant properties. Site-directed mutagenesis experiments indicated that 1 acts at an allosteric site involving residue F77. Thus, 1 is a promising drug because the M1 activation may offer disease-modifying properties that could address and reduce most of AD hallmarks. BPH is an enlargement of the prostate caused by increased cellular growth. Blockade of α1-ARs is the predominant form of medical therapy for the treatment of the symptoms associated with BPH. α1-ARs are classified into three subtypes. The α1A- and α1D-AR subtypes are predominant in the prostate, while α1B-ARs regulate the blood pressure. Herein, we report the synthesis of quinazoline-derivatives obtained replacing the piperazine ring of doxazosin and prazosin with (S)- or (R)-3-aminopiperidine. The presence of a chiral center in the 3-C position of the piperidine ring allowed us to exploit the importance of stereochemistry in the binding at α1-ARs. It turned out that the S configuration at the 3-C position of the piperidine increases the affinity of the compounds at all three α1-AR subtypes, whereas the configuration at the benzodioxole ring of doxazosin derivatives is not critical for the interaction with α1-ARs.

New biosensor applications of surface plasmon and hydrogel optical waveguide spectroscopy

Rapid and sensitive detection of chemical and biological analytes becomes increasingly important in areas such as medical diagnostics, food control and environmental monitoring. Optical biosensors based on surface plasmon resonance (SPR) and optical waveguide spectroscopy have been extensively pushed forward in these fields. In this study, we combine SPR, surface plasmon-enhanced fluorescence spectroscopy (SPFS) and optical waveguide spectroscopy with hydrogel thin film for highly sensitive detection of molecular analytes.rnrnA novel biosensor based on SPFS which was advanced through the excitation of long range surface plasmons (LRSPs) is reported in this study. LRSPs are special surface plasmon waves propagating along thin metal films with orders of magnitude higher electromagnetic field intensity and lower damping than conventional SPs. Therefore, their excitation on the sensor surface provides further increased fluorescence signal. An inhibition immunoassay based on LRSP-enhanced fluorescence spectroscopy (LRSP-FS) was developed for the detection of aflatoxin M1 (AFM1) in milk. The biosensor allowed for the detection of AFM1 in milk at concentrations as low as 0.6 pg mL-1, which is about two orders of magnitude lower than the maximum AFM1 residue level in milk stipulated by the European Commission legislation.rnrnIn addition, LRSPs probe the medium adjacent to the metallic surface with more extended evanescent field than regular SPs. Therefore, three-dimensional binding matrices with up to micrometer thickness have been proposed for the immobilization of biomolecular recognition elements with large surface density that allows to exploit the whole evanescent field of LRSP. A photocrosslinkable carboxymethyl dextran (PCDM) hydrogel thin film is used as a binding matrix, and it is applied for the detection of free prostate specific antigen (f-PSA) based on the LRSP-FS and sandwich immunoassay. We show that this approach allows for the detection of f-PSA at low femto-molar range, which is approximately four orders of magnitude lower than that for direct detection of f-PSA based on the monitoring of binding-induced refractive index changes.rnrnHowever, a three dimensional hydrogel binding matrix with micrometer thickness can also serve as an optical waveguide. Based on the measurement of binding-induced refractive index changes, a hydrogel optical waveguide spectroscopy (HOWS) is reported for a label-free biosensor. This biosensor is implemented by using a SPR optical setup in which a carboxylated poly(N-isoproprylacrylamide) (PNIPAAm) hydrogel film is attached on a metallic surface and modified by protein catcher molecules. Compared to regular SPR biosensor with thiol self-assembled monolayer (SAM), HOWS provides an order of magnitude improved resolution in the refractive index measurements and enlarged binding capacity owing to its low damping and large swelling ratio, respectively. A model immunoassay experiment revealed that HOWS allowed detection of IgG molecules with a 10 pM limit of detection (LOD) that was five-fold lower than that achieved for SPR with thiol SAM. For the high capacity hydrogel matrix, the affinity binding was mass transport limited.rnrnThe mass transport of target molecules to the sensor surface can play as critical a role as the chemical reaction itself. In order to overcome the diffusion-limited mass transfer, magnetic iron oxide nanoparticles were employed. The magnetic nanoparticles (MNPs) can serve both as labels providing enhancement of the refractive index changes, and “vehicles” for rapidly delivering the analytes from sample solution to an SPR sensor surface with a gradient magnetic field. A model sandwich assay for the detection of β human chorionic gonadotropin (βhCG) has been utilized on a gold sensor surface with metallic diffraction grating structure supporting the excitation of SPs. Various detection formats including a) direct detection, b) sandwich assay, c) MNPs immunoassay without and d) with applied magnetic field were compared. The results show that the highly-sensitive MNPs immunoassay improves the LOD on the detection of βhCG by a factor of 5 orders of magnitude with respect to the direct detection.rn