Orientador: Marcos Hikary Toyama / Resumo: A giroxina é a principal serino protease encontrada no veneno total de Crotalus durissus terrificus. As serino proteases de veneno de serpentes são proteínas que apresentam uma grande similaridade estrutural com a trombina humana incluindo a alta conservação do sitio catalítico. Os resμltados já obtidos pelo nosso grupo revelaram a capacidade dos flavonoides glicosilados de Laguncularia racemosa em reduzir de forma significativa a atividade enzimática das trombinas humanas. A identificação destas molécμlas revelou que dois flavonoides glicosilados (quercetina-3-O-arabinosidio (QAra) e quercetina-3-O-ramnosidio (QRham)) respondem pela inibição da trombina (Rodrigues et al., 2015). Sabe-se que a soroterapia antiveneno não consegue neutralizar de forma eficiente a ação farmacológica das serino proteases e consequentemente não é capaz de neutralizar os danos homeostáticos e teciduais. Neste trabalho, uma nova serino protease foi isolada do veneno total de Crotalus durissus terrificus (cdtsp-2) através de quatro estapas de separação, usando a superdex 75 com uma fase móvel de bicarbonato de amônia pH 8, a cdtsp-2 Estas duas frações foram então fracionadas em coluna de troca iônica em DEAE para separação das principais frações e posteriormente fracionadas em CLAE de fase reversa para confirmar o grau de homologia molecular e em todas as etapas cromatográfica foram acompanhadas pelos ensaios enzimáticos em BApNA. A nova serino protease denominada como Cdtsp-2 (Crotalus durissus terr... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Gyroxin is the major serine protease present in Crotalusdurissusterrificus venom. Serine proteases from snake venom are proteins, which exhibit a great structural similarity to hμMan thrombin including the catalytic site conservation. The resμlts already obtained by our group revealed the glycosylated flavonoidsability of Laguncμlariaracemosato significantly reduce the enzymatic activity of hμMan thrombins. The molecμles identification reveals two glycosylated flavonoids (quercetin-3-O-arabinosidiμM (QAra) and quercetin-3-O-rhamnosidiμM(QRham)), which inhibit thrombin (Rodrigues et al., 2015). Antivenomserotherapycan not efficiently neutralize the serine proteases pharmacological action and is not able to neutralize homeostatic and tissue damage. In this work, a new serine protease was isolated from Crotalusdurissusterrificusvenom (cdtsp-2) by throμgh four separation stages , using superdex 75 with a mobile phase of ammoniμM bicarbonate pH 8. These two fractions were fractionated on ion exchange colμMn in DEAE for separation of the major fractions and subsequently fractionated in reverse phase CLAE to confirm the degree of molecμlar homology.All chromatographic steps were monitored by the enzymatic assays in BApNA. The new serine protease called cdtsp-2 (Crotalusdurissusterrificus serine protease 2) is an unpublished protein, with a molecμlar mass of 27 kDa and its N-terminal sequence showed structural differences when compared to the gyroxin of Crotalusdurissusterrificus. Re... (Complete abstract click electronic access below) / Mestre

Le syndrome de détresse respiratoire aiguë (SDRA) est caractérisé par des dommages au niveau de la barrière alvéolo-capillaire, résultant en la formation d’un œdème pulmonaire et une réponse inflammatoire exacerbée. Sans résolution rapide de ces paramètres, le syndrome progresse vers le développement de fibrose menant à l’insuffisance respiratoire. Or, il a été établi que la réparation de l’épithélium alvéolaire est une étape cruciale pour la résolution du SDRA. Une meilleure compréhension des mécanismes de réparation de l’épithélium alvéolaire est donc nécessaire afin de proposer de nouvelles thérapies pour le SDRA, pour lequel aucun traitement efficace n’existe. Il a été montré que les mécanismes de réparation sont régulés par des protéines membranaires, non seulement par les récepteurs aux facteurs de croissance et les intégrines, mais également par les canaux ioniques, en particulier les canaux potassiques. L’objectif principal de cette étude était donc de caractériser l’impact de la modulation des canaux potassiques KCa3.1 et KvLQT1 dans la résolution du SDRA. Dans un premier temps, nos résultats ont montré le rôle coopératif du canal potassique KCa3.1, de la matrice extracellulaire et de l’intégrine-β1 dans les processus de réparation de l’épithélium alvéolaire in vitro. Nous avons montré que la matrice de fibronectine et le KCa3.1 étaient impliqués dans la migration et dans la réparation de monocouches de cellules alvéolaires de cultures primaires de rat. Dans un deuxième temps, nous avons étudié l’impact de la modulation du canal potassique KvLQT1 dans certains aspects physiopathologiques du SDRA à l’aide de modèles in vivo. Nous avons montré que KvLQT1 n’était pas seulement impliqué dans les mécanismes de réparation de l’épithélium alvéolaire, mais également dans la résorption de l’œdème pulmonaire et la résolution de la réponse inflammatoire. Nos résultats démontrent que les canaux potassiques, tels que KCa3.1 et KvLQT1, pourraient être identifiés en tant que cibles thérapeutiques potentielles pour le SDRA. / Acute respiratory distress syndrome (ARDS) is characterized by alveolar-capillary barrier damage, resulting in the formation of pulmonary oedema and an exacerbated inflammatory response. Without rapid recovery of these parameters, there is a gradual development of fibrosis, leading to respiratory failure. It has been established that alveolar regeneration is a critical step for the resolution of ARDS. A better understanding of alveolar epithelial repair mechanisms is hence necessary to identify new therapies for ARDS, for which no effective treatment exist. It has been shown that repair mechanisms are regulated by membrane proteins, not only by growth factor receptors and integrins, but also by ion channels, in particular potassium channels. Therefore, the main objective of this study was to characterize the impact of KCa3.1 and KvLQT1 potassium channels modulation in the resolution of ARDS. First, our results have shown the cooperative role of the potassium channel KCa3.1, the extracellular matrix and the β1-integrin in alveolar epithelial repair processes in vitro. We have shown that the fibronectin matrix and KCa3.1 are involved in the migration and repair of primary cultures of rat alveolar cell monolayers. Our data also revealed a putative relationship between Kca3.1 and the β1-integrin. Second, we studied the impact of KvLQT1 potassium channel modulation on ARDS pathophysiological aspects with in vivo models. We showed that KvLQT1 was not only involved in alveolar epithelial repair, but also in the resolution of pulmonary oedema and inflammatory response. Taken together, our data demonstrate that potassium channels, such as KCa3.1 and KvLQT1, may be identified as potential therapeutic targets for the resolution of ARDS.

Hintergrund: Die bakterielle Meningitis hat eine Letalität von 10-20%. Das Hirnödem stellt bei ca. 14 % der Erkrankten eine prognosebestimmende Komplikation dar. Ein aktueller Forschungsansatz umfasst die Bedeutung der Aquaporine für die Entwicklung, Aufrechterhaltung und Resorption der verschiedenen Hirnödemformen, insbesondere des zytotoxischen und des vasogenen Hirnödems. In dieser Arbeit wird untersucht, ob Aquaporin 1 und Aquaporin 4 im Liquor von Patienten mit bakterieller und viraler Meningitis, im Vergleich zu einer gesunden Kontrollgruppe, nachweisbar sind. Zudem sollte geklärt werden, ob sich hieraus eine differenzialdiagnostische Einordnung ergibt und sich Rückschlüsse auf das Ausmaß eines Hirnödems und das Outcome schließen lassen. Methode: Aquaporin 1 und 4 wurde im Liquor und im Serum von Patienten mit bakterieller (nCSF = 35 , nSerum = 20) und viraler  (nCSF = 22) Meningitis sowie in einer Kontrollgruppe (nCSF = 27 , nSerum = 12) mittels eines (kommerziell erhältlichen) ELISAs bestimmt. Klinische Daten und Routinelaborparameter wurden verglichen und in Korrelation zu den Aquaporinkonzentrationen gesetzt. Ergänzend wurde bei einer Untergruppe der Patienten mit bakterieller Meningitis (n = 8) eine neuropsychologische Testung durchgeführt. Ergebnisse: Aquaporin 1 und 4 ließen sich in allen Gruppen nachweisen, ca. 40% der Aquaporin 4 Konzentrationen lagen unterhalb der Nachweisgrenze des ELISAs. Im Gruppenvergleich aller drei Gruppen unterschieden sich die Aquaporin 1-Konzentrationen (p = 0,0001) und die Aquaporin 4-Konzentrationen (p = 0,035) im Liquor signifikant voneinander. In der Gruppe der Patienten mit bakterieller Meningitis ließ sich eine negative Korrelation zwischen Aquaporin 1 und 4 im Liquor feststellen (r = - 0,519, p = 0,002). Aussagekräftige Korrelationen der klinischen Daten, der liquor- und laborchemischen Parameter sowie der neuropsychologischen Testergebnisse zu den Aquaporin 1- und Aquaporin 4-Konzentrationen fanden sich nicht.  Diskussion: In dieser Arbeit konnte erstmalig gezeigt werden, dass Aquaporin 1 und Aquaporin 4 im Liquor (und Serum) von Patienten mit einer bakteriellen und viralen Meningitis sowie in einer Kontrollgruppe nachweisbar sind. Für Aquaporin 1 und Aquaporin 4 im Liquor fanden sich signifikante Unterschiede im Vergleich aller Gruppen im Kruskal-Wallis-Test. Rückschlüsse bezüglich einer differenzial-diagnostischen Einordnung zur viralen Meningitis konnten nicht gezogen werden. Aussagen zur Schwere eines Hirnödems und zur Prognose können mit den vorliegenden Daten nicht getroffen werden. Der Ursprung der gemessenen Aquaporine bei Patienten mit Meningitis lässt sich in dieser Arbeit nicht abschließend klären und bedarf weiterer Grundlagenforschung.

<p>Dijabetesna retinopatija je među vodećim uzročnicima stečenog slepila, kako u razvijenim zemljama, tako i zemljama u razvoju. Dijabetesna retinopatija je jedna od<br />najče&scaron;ćih komplikacija Dijabetes Mellitus-a. U sklopu dijabetesne retinopatije jedan od najranijih razloga koji dovodi do pada vidne o&scaron;trine je dijabetični makularni edem (DME). Pad vidne o&scaron;trine kod pacijenata sa dijabetesom naru&scaron;ava njihov kvalitet života i umanjuje radnu sposobnost. Dosada&scaron;nji oblik lečenja laserfotokoaguacijom makule, nije dao zadovoljavajuće rezultate. U novije vreme sve vi&scaron;e je zastupljeno farmakolo&scaron;ko lečenje edema koje podrazumeva intrvitrealnu aplikaciju lekova iz grupe inhibitora vaskularnog endotelnog faktora rasta (VEGF inhibitori), koji dovodi do stabilizacije zidova krvnih sudova.&nbsp;<br />Cilj ove studije je da se ispita efikasnost lečenja DME uz pomoć intravitrealno aplikovanih lekova iz grupe inhibitora vaskularnog endotelnog faktora rasta u odnosu na konvencionalno do sada priznato lečenje laserfotokogulacijom makule.&nbsp;<br />Efikasnost lečenja je procenjivana na dva načina: anatomski, na osnovu smanjenja centralne makularne debljine izražene u &mu;m, merene metodom optičke koherentne tomografije, i funkcionalno, na osnovu pobolj&scaron;anja vidne o&scaron;trine koja je izražavana u log MAR jedinicama. U ovoj prospektivnoj, randomiziranoj kliničkoj studiji sa minimumom praćenja od 6 meseci, u eksperimentalnoj grupi tretiran je 51 pacijent,<br />odnosno 84 oka aplikacijom bevacizumaba (anti VEGF agens) u dozi od 1,25 mg, sa ili bez dodatnog laser tretmana.&nbsp;<br />Uz prosečno 2,46 inekcije postignuta je prosečna redukcija centralne makularne debljine od 139,15 &mu;m.&nbsp; Dobijene vrednosti su nakon svake aplikovane doze su značajno bolje u odnosu na početnu. Edemi sa većom centralnom makularnom debljinom su zahtevali tretman sa većim brojem inekcija. Kod većih edema je postignuta i veća redukcija centralne makularne debljine. U odnosu na vidnu o&scaron;trinu u eksperimentalnoj grupi postignuto je pobolj&scaron;anje od 0,135 log MAR jedinica. Efekat lasera kao samostalne terapije u kontrolnoj grupi (50 pacijenata, 92 oka) nije bio<br />značajan ni u pogledu smanjenja centralne makularne debljine kao ni na osnovu pobolj&scaron;nja vidne o&scaron;trine. Tretman bevacizumabom samostalno ili u kombinaciji sa laserom je efikasniji u tretmanu DME u odnosu na konvencionalni tretman laserfotokoaguacijom koji potvrđeno dovodi do stabilizacije stanja. Značaj ove studije je potvrda efikasnosti i bezbednosti jednog novog oblika lečenja koji samostalno ili u kombinaciji sa laser tretmanom predstavlja novi protokol lečenja dijabetičnog makularnog edema.</p> / <p>Diabetic retinopathy is among the leading causes of acquired blindness in developed countries, as well as in developing countries. Diabetic retinopathy is one of the most frequent Diabetes Mellitus complications. Within diabetic retinopathy, diabetic macular edema (DME) is one of the earliest causes of the loss of visual acuity. Impaired vision causes decline in life quality in diabetic patients and it decreases their<br />working ability. Up to this date, laser photocoagulation treatment has not given<br />satisfactory results. Recently, new promising treatment forms have emerged, including the intravitreal application of vascular endothelium growth factor (VEGF inhibitors), which lead to stabilization of the vessel wall. The aim of this study is to evaluate the efficacy of DME treatment consisting of intravitreal&nbsp; VEGF inhibitor application alone or as a part of combined treatment (intravitreal VEGF inhibitor plus laser photocoagulation) compared with conventional laser treatment alone. The effect of treatment was evaluated according to morphological parameters by measuring central macular thickness (CMT) in &mu;m with optical coherence tomography, and according to functional parameter by visual acuity in log MAR scale. In this prospective randomized clinical trial, with minimum follow up of 6 months, in experimental group 51 patient, or 84 eyes were treated with bevacizumab (VEGF inhibitor) in 1.25 mg dosage, alone or in combination with laser. The mean reduction in was 139.15 &mu;m, which was achieved with 2.46 doses on average. The difference between the final and initial CMT values after each dos age was tatistically significant.<br />Edemas with high central macular thickness required high number of intraviteal<br />aplicatons and the reduction was higher. In our study, mean visual acuity improved significantly in 0.135 log MAR. In control group (50 patient, 92 eyes) treated with laserphotocolagulation alone, the effect on visual acuity and central acular thickness was not statistically significant. The treatment with bevacizumab alone or in combined<br />treatment is more effective in treating DME than conventional macular laser treatment alone, from both - anatomical and functional perspective. The importance of this study is confirmation of the efficacy and safety of a new form of treatment and the introduction of a new protocol for the treatment of diabetic macular edema.</p>

<p>Cilj ovog istraživanja je da se ispita uticaj centralne debljine makularne regije (CMT) i prisustva subretinalne tečnosti na vidnu o&scaron;trinu (VA) kod pacijenata obolelih od dijabetesnog makularnog edema, kao i uticaj očuvanosti kontinuiteta spoja spolja&scaron;njeg i unutra&scaron;njeg segmenta fotoreceptora (IS/OS &ndash; kompleks) i spolja&scaron;nje granične membrane (ELM) kao prognostičkih faktora u pobolj&scaron;anju vidne o&scaron;trine nakon primenjene terapije kod pacijenata obolelih od dijabetesnog makularnog edema (DME). Materijal i metode: u ovu retrospektivno prospektivnu kliničku studiju nasumično je uključeno 100 pacijenata koji su podeljeni u dve grupe. Grupu A &ndash; prospektivni deo studije je činilo 50 pacijenata (50 očiju) kod kojih je dijagnostikovan dijabetesni makularni edem i kod kojih je inidikovana primena terapije, laserftotkogaulacije i/ili anti-VEGF terapije (bevacizumab). Grupu B &ndash; retrospektivnu grupu je činilo 50 pacijenata (58 očiju) koji su prethodno lečeni od dijabetesnog makularnog edema primenom laserfotokoagulacije i/ili anti-VEGF terapije (bevacizumab). Nakon kompletnog oftalmolo&scaron;kog pregleda koji se sastojao od određivanja vidne o&scaron;trine (optotipima po Snellenu), biomikroskopije, merenja intraokularnog pritiska i pregleda očnog dna &ndash; fundusa primenom panfundoskopa izvr&scaron;ena je optička koherentna tomografija u svih pacijenata (primenom aparata Stratus&reg; OCT, Carl Zeiss, Meditec i Copercnicus&reg; Optopol). Analiza OCT snimka, je obuhvatila određivanje centralne debljine makule (CMT), prisustva subretinalne tečnosti kao i procenu stanja očuvanosti kontinuiteta spoja spolja&scaron;enjeg i unutra&scaron;njeg segmenta fotoreceptora (IS/OS kompleks) i očuvanost kontinuiteta spolja&scaron;nje granične membrane (ELM). CMT je izračunat primenom softvera OCT aparata i izražen kao srednja vrednost za svih 9 ETDRS polja. Prisutvno subretinalne tečnosti je klasifikovano kao pozitivno ukoliko je identifikovano makar u jednom preseku OCT tomograma .Očuvanost kontinuiteta IS/OS kompleksa i ELM je analizirana u svakom pojedinačnom snimku i podeljena u 3 kategorije. Prva &ndash; ukoliko je očuvano u svim presecima, druga &ndash; ukoliko je očuvano samo u pojedinim presecima i treća &ndash; ukoliko se IS/OS kompleks i ELM nisu mogli identifikovati na nalazu OCT tomograma. Rezultati ukazuju da prisustvo subretinalne tečnosti pre primenjene terapije nema statistički značajnog uticaja na pobolj&scaron;anje vidne o&scaron;trine nakon primenjene terapije u pacijenata grupe A (pA=0,915), a statistička značajnost nije potvrđena ni kod pacijenata koji su prethodno tretirani od DME &ndash; grupa B (pB=0,772). Srednja vrednosti CMT i VA u pacijaneta grupe A iznosila je 474&mu;m&plusmn;140,67&mu;m odnosno 0.25&plusmn;0.20. Nakon primenjene terapije srednja vrednost vidnih o&scaron;trina iznosila je 0.41&plusmn;0.25, dok su vrednosti srednje vrednosti CMT iznosile 343.68&mu;m&plusmn;99.03&mu;m. Potvrđeno je statistički značajno pobolj&scaron;anje vidne o&scaron;trine nakon primenjene terapije (pVA=0,0001) i statistički značajno smanjenje centralne debljine makule (pCMT=0,0001). Korelacija VA sa vrednostima CMT pre primenjene terapije pokazuje statističku značajnost sa negativnom korelacijom (r=-0,391; p=0,005) dok se nakon primenjene terapije ne uočava statistički značajna korelacija (r=-0,047; p=0,746). Analizom vrednosti CMT pre primenjene terapije sa vrednostima VA nakon terapije se uočava statistički značajna negativna korelacija, odnosno veće vrednosti CMT pre primenjene terapije ograničavaju pobolj&scaron;anje vidne o&scaron;trine nakon primenjene terapije (r=-0,393; p=0,005). Evaluacija OCT tomograma, pre primenjene terapije, u pacijenata grupe A utvrđen je u potpunosti očuvan kontinuitet IS/OS kompleksa i ELM u svim presecima u 23 odnosno 27 očiju, u pojedinim presecima u 18 odnosno 16 očiju, i nije mogao biti identifikovan u 9 odnosno 7 očiju. U pacijenata grupe A nakon primenjene terapije uočava se statistički značajno pobolj&scaron;anje vrednosti VA u zavisnosti od očuvanosti kontinuiteta IS/OS kompleksa (F=5,550, p=0,007) i ELM (F=5,428, p=0,008). Univarijantna odnosno multivarjiantna analiza podataka za granične vrednosti vidnih o&scaron;trina od 0,1 i koraka pobolj&scaron;anja od 0,1 ukazuje na statističku značajnost prediktora IS/OS kompleksa (p=0,012 i p=0,032) i ELM (p=0,003 i p=0,018) u pobolj&scaron;anju vrednosti vidnih o&scaron;trina nakon primenjene terapije. Pacijenti sa očuvanim kontinuitetom IS/OS kompelsa u svim presecima imaju 9,5 puta (OR=9,500 ) veću &scaron;ansu za pobolj&scaron;anje VA nakon primenjene terapije u odnosu na pacijente gde kontinuitet IS/OS kompleksa nije uočljiv. Pacijenti sa očuvanim kontinuitetom IS/OS kompleksa u pojedinim presecima imaju 7 puta veću &scaron;ansu (OR=7,000) za pobolj&scaron;anje vidne o&scaron;trine nakon terapije u poređenju sa onima kod kojih IS/OS nije uočljiv. Pacijenti sa očuvanim kontinuitetom ELM u svim presecima imaju 34,5 puta (OR=34,500 ) veću &scaron;ansu za pobolj&scaron;anje vidne o&scaron;trine u odnosu na pacijente gde ELM nije uočljiv. Pacijenti sa očuvanim kontinuitetom ELM u pojedinim presecima imaju 18 puta veću &scaron;ansu (OR=18,000) za pobolj&scaron;anje VA nakon terapije u odnosu na one kod kojih ELM nije uočljiv. Pored statistički značajnog uticaja očuvanosti kontinuiteta IS/OS kompleksa i ELM na pobolj&scaron;anje vrednosti vidnih o&scaron;trina nakon primenjene terapije, uočava se i pozitvna korelacija između vidnih o&scaron;trina pre i nakon terapije (r=0,869; p=0,0001). U pacijenata grupe B srednja vrednost CMT odnosno VA iznosila je 253,72&mu;m&plusmn;75,27&mu;m odnosno 0,68&plusmn;0,29. Postoji statistički značajna razlika u vrednostima VA u odnosu na očuvanost kontinuiteta IS/OS kompleksa (F=107,913, p=0,0001) i ELM (F=25,619, p=0,0001). Poređenjem vrednosti parametara za obe posmatrane grupe uočava se statistički značajna razlika u vrednostima CMT koje su bile manje u grupi B (t=5,355, p=0,0001) i srednjim vrednostima VA ( t=5,137, p=0,0001) koje su bile veće u grupi B. Analizom očuvanosti kontinuiteta IS/OS kompleksa (&chi;2=0,119, p=0,730) i ELM (&chi;2=2,957, p=0,085) ne uočava se statistički značajna razlika. Zaključak: Odnos vidnih o&scaron;trina sa centralnom debljinom makule prikazuje različite vrednosti vidnih o&scaron;trina za iste vrednosti centralne debljine makule. Značajan uticaj na vidnu o&scaron;trinu pacijenata obolelih od DME ima očuvanost integriteta spolja&scaron;nje granične membrane (ELM) i spoja unutra&scaron;njeg i spolja&scaron;njeg segmenta fotoreceptora (IS/OS kompleks) evaluiranih na osnovu OCT snimka &ndash; tomograma. Očuvanost integriteta ELM i IS/OS kompleksa u svim presecima na OCT tomogramu pre primenjene terapije u pacijenta sa DME se mogu smatrati pozitivnim prognostičkim faktorom u pobolj&scaron;anju vidne o&scaron;trine nakon primenjene terapije. U pacijenata kod kojih je kontinuitet ELM i IS/OS kompleksa očuvan u svim pravcima vrednost CMT pre primenjene terapije nema uticaj na pobolj&scaron;anje vidne funkcije nakon terapije. Integritet IS/OS kompleksa i ELM ima pozitivnu korelaciju sa vidnom o&scaron;trinom bez obzira na vrstu primenjene terapije, anti-VEGF odnosno laserfotokoagulacije. Prisustvo subretinalne tečnosti ne utiče na vidnu o&scaron;trinu pacijenata obolelih od DME. Vrednosti VA pre terapije utiču na pobolj&scaron;anje vidne o&scaron;trine nakon terapije.</p> / <p>The aim of this study was to investigate the influence of the central macular thickness (CMT) and the presence of sub retinal fluid on visual acuity (VA) in patients with diabetic macular edema, as well as the impact of preservation and continuity of the photoreceptor inner/outer segment junction (IS / OS - complex ) and external limiting membrane (ELM) as a prognostic factor in improving visual acuity after the applied therapy in patients with diabetic macular edema (DME). Materials and Methods: this retrospective - prospective randomized clinical study included 100 patients who were divided into two groups. Group A - a prospective part of the study, consisted of 50 patients (50 eyes), with the diagnosis of diabetic macular edema in which laser photocoagulation and / or anti-VEGF therapy (bevacizumab) was indicated. Group B - retrospective group, consisted of 50 patients (58 eyes), who were previously treated for diabetic macular edema either with laser photocoagulation and / or anti-VEGF therapy (bevacizumab). After complete ophthalmologic examination, which consisted of the determination of visual acuity (measured with Snellen charts), biomicroscopy, intraocular pressure measurement and inspection of the fundus, optical coherence tomography was performed in all patients (using the Stratus&reg; OCT, Carl Zeiss Meditec and Copercnicus&reg; Optopol). Analysis of OCT image, included the determination of the central macular thickness (CMT), presence of sub retinal fluid, as well as an assessment of the preservation of the continuity of the photoreceptor inner/outer segment junction (IS/OS - complex) and external limiting membrane (ELM). CMT is calculated using software of the OCT apparatus and expressed as the mean value for all 9 ETDRS fields. Presence of sub retinal fluid is classified as positive if it is identified in at least one cross-section of OCT tomogram. Preserved continuity of IS / OS complex and ELM is analyzed in each individual OCT cross-section image and divided into 3 categories. First - if it is preserved in all cross sections images, the second - if it is preserved only in certain sections and the third - if the IS / OS complex and ELM were not able to identify in OCT tomograms. The results indicate that the presence of sub retinal fluid before the applied therapy has no statistically significant effect on improving visual acuity after the applied therapy in patients of group A (pA = 0.915), and statistical significance was not also confirmed in any of the patients who were previously treated by DME - Group B (pB = 0.772). Mean CMT and VA values of patients in group A was 474&mu;m &plusmn; 140,67&mu;m and 0.25 &plusmn; 0.20. After receiving therapy mean visual acuity was 0.41 &plusmn; 0.25, while the value of the mean CMT was 343.68&mu;m&plusmn; 99.03&mu;m. Significant improvement in visual acuity was achieved after the treatment in group A (pVA = 0.0001) together with statistically significant reduction in central macular thickness (pCMT = 0.0001). Correlation of VA with the values of CMT before applied therapy shows statistically significant negative correlation (r = -0.391; p = 0.005), while after the applied therapy statistical significance was not observed (r = -0.047; p = 0.746). Analyzing the values of CMT before the applied therapy with the values of VA after the treatment statistically significant negative correlation was observed, higher values of CMT before the applied therapy restrict visual acuity improvement after the applied therapy (r = -0.393; p = 0.005). Analyzing OCT tomograms in the patients in group A, before the applied therapy, fully preserved continuity of IS/OS complex and ELM in all the sections was found in 23 and 27 of the eyes, in certain sections in 18 and 16 of the eyes, and could not be identified in 9 and 7 eyes. Statistically significant improvement in VA, after the applied therapy, in patients in group A is observed, depending on the preservation of continuity of IS/OS complex (F = 5.550, p = 0.007) and ELM (F = 5.428, p = 0.008). Univariate and multivariate analysis with cut off VA value of 0.1 and step improvements of 0.1 points to statistically significant predictor of IS/OS complex (p = 0.012 and p = 0.032) and ELM (p = 0.003 and p = 0.018) in improving the VA after the applied therapy. Patients with preserved continuity of IS/OS complex in all sections are 9.5 times (OR = 9.500) more likely to improve the VA after receiving therapy compared to patients where continuity of IS/OS complex is not noticeable. Patients with preserved continuity of IS/OS complex in the some sections are 7 times more likely (OR = 7.000) for the improvement of visual acuity after treatment compared to those in which the IS/OS is not detectable. Patients with preserved continuity of ELM in all sections are 34.5 times (OR = 34,500) a greater chance to improve visual acuity compared to patients where ELM is not apparent. Patients with preserved continuity of ELM in the some sections are 18 times more likely (OR = 18,000) to improve the VA after treatment compared to those in which the ELM is not apparent. In addition to statistically significant impact of preservation of continuity of IS/OS complex and ELM for VA improvement after the treatment, statistically significant positive correlation between visual acuity before and after treatment (r = 0.869; p = 0.0001) was observed. In Group B patients, the mean CMT and VA value was 253,72&mu;m&plusmn;75,268&mu;m and 0.68 &plusmn; 0.29. There is a statistically significant difference in the VA values compared to the preservation of continuity of IS/OS complex (F = 107.913, p = 0.0001) and ELM (F = 25.619, p = 0.0001). Comparing the values of parameters for both groups, statistically significant difference in CMT values and mean VA was observed. CMT values were lower (t = 5.355, p = 0.0001) while VA values were higher (t = 5.137, p = 0.0001), in group B. The analysis of preservation of continuity of IS/OS complex (&chi;2 = 0.119, p = 0.730) and ELM (&chi;2 = 2.957, p = 0.085) did not show a statistically significant difference. Conclusion: The relationship of visual acuity with central macular thickness shows the different levels of visual acuity for the same value of the central macular thickness. A significant impact on VA in patients with DME has maintained integrity of the external limiting membrane (ELM) and the photoreceptors inner/outer segments junction (IS/OS complex) evaluated on the basis of OCT - tomograms. Preservation of the integrity of the ELM and IS/OS complex in all sections of the OCT tomogram before applied therapy in patients with DME can be considered a positive prognostic factor in improving visual acuity after receiving therapy. In patients with preserved continuity of ELM and IS/OS complex in all sections before applied therapy the CMT value has no effect on the improvement of visual function after treatment. Regardless of the type of applied therapy, anti-VEGF and/or laser photocoagulation preserved integrity of IS/OS complex and ELM has a positive correlation with visual acuity. The presence of sub retinal fluid does not affect the visual acuity in patients with DME. The values of VA before treatment influence the improvement of visual acuity after treatment.</p>

Descreve-se os achados epidemiológicos e clínico-patológicos das clostridioses diagnosticadas no Setor de Patologia Veterinaria da Universidade Federal do Rio Grande do Sul no período 1996-março/2010. Este estudo incluiu uma pesquisa retrospectiva nos arquivos do SPV e uma etapa prospectiva, a qual também teve o objetivo de desenvolver exames complementares específicos para cada clostridiose. As clostridioses mais prevalentes foram tétano (em equinos, bovinos, ovinos e caprinos), botulismo (em bovinos, suínos e aves) e enterotoxemia (em caprinos). Também houve casos de edema maligno em equinos, bem como de carbúnculo sintomático e hemoglobinúria em bovinos. Adicionalmente, foram coletadas amostras de soro sanguíneo de animais afetados por tétano, em diferentes estágios de evolução da doença, para subsequente inoculação em camundongos (testes de bioensaio) e comprovação da técnica no diagnóstico da enfermidade. / This study describes the epidemiological and clinicopathological findings of clostridial diseases diagnosed in the 1996-March, 2010 period in the Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul (SPV_UFRGS). A retrospective survey in the files of SPV was complemented with a prospective phase, which also aimed developing complementary diagnostic tests of clostridiosis. The most prevalent clostridiosis were tetanus (in horses, cattle, sheep and goats), botulism (in cattle, pigs and birds), and enterotoxemia in goats. There also were cases of malignant edema in horses, blackleg and bacillary hemoglobinuria in cattle. In addition, blood serum samples from animals affected by tetanus on different stages of the disease evolution were applied in mice bioassay, as a complementary diagnosing test for the disease.

A exodontia de terceiros molares inclusos produz um grau significativo de traumatismo para os tecidos moles e estruturas ósseas da cavidade oral, consequentemente resultando em uma reação inflamatória. Esta reação gera diversos sinais e sintomas clínicos pós operatórios como: dor, edema e limitação de abertura bucal que fazem parte da resposta inflamatória reparadora, mas que se exacerbada pode levar a grandes desconfortos. O objetivo do presente estudo foi verificar comparativamente a piezocirurgia e a técnica convencional rotatória quanto a variabilidade dos principais parâmetros clínicos, sistêmicos e locais, no pré, trans e no pós operatório da exodontias de terceiros molares inferiores. Foi realizado um estudo clínico randomizado, cruzado em 20 pacientes saudáveis com necessidade de exodontia de terceiros molares inclusos e/ou impactados bilateralmente de mesma dificuldade cirúrgica. O lado a ser operado foi alocado randomicamente para técnica ultrassônica ou rotatória. Foram analisadas as alterações hemodinâmicas, pressão arterial, frequência cardíaca e saturação de oxigênio pré, trans e pós operatórias. Os desfechos locais foram coletados no dia do procedimento e 24 horas após a cirurgia, nesse contexto foi investigada a máxima abertura bucal em milímetros e o edema facial em pontos anatômicos pré estabelecidos. A análise de dor foi registrada pelo próprio paciente por meio da escala visual analógica em ficha padronizada, assim como a quantidade de analgésicos consumida e os níveis de ansiedade. Os resultados mostraram que o tempo trans operatório pelo uso da piezocirurgia foi maior. Não houve grande variabilidade nos parâmetros hemodinâmicos. A técnica ultrassônica provocou limitação de abertura de boca semelhante à técnica convencional. Gerou menor edema pós operatório em uma das dimensões lineares. Não promoveu níveis menores de dor pós operatória, além disso pode aumentar o grau de ansiedade do paciente. Concluímos que para as osteotomias nas exodontias de terceiros molares inferiores, a piezocirurgia mostrou ser comparável a técnica convencional. O emprego de uma ou outra técnica deve ser uma opção pessoal do cirurgião. / The removal of third molars leads a significant degree of trauma to soft tissues and bone structures of oral cavity, consequently results in an inflammatory reaction. This reaction generates several clinical signs and post-operative symptoms such as: pain, edema and oral aperture limitation that are part of the inflammatory response. If exacerbated, this response can lead to a great discomfort. The aim of this study was to compare, systemic and local parameters variations, between the piezosurgery and the conventional rotational technique for third molar removal. A randomized, crossover clinical trial was carried out in 20 healthy patients with the necessity for bilateral impacted third molars extraction with the same surgical difficulty. The side to be operated was allocated randomly for ultrasonic or conventional rotatory technique. Hemodynamic variations as: blood pressure, heart rate and oxygen saturation were analyzed in different moments. The local outcomes: maximum oral aperture and facial edema were measured on the day of the procedure and 24 hours after the surgery. Post-operative pain scores were recorded by the patient using a visual analogical scale in standardized diary, as well as anxiety levels and the number of painkillers intake. The results showed that the trans-operative period with the use of piezosurgery was higher. There was no great variability at the hemodynamic parameters. The ultrasonic technique caused a mouth-aperture limitation like the conventional technique and it generated minor post-operative edema in one of the linear dimensions. It did not promote minor levels of post-operative pain, moreover it can increase the anxiety level of the patient. We concluded that for osteotomies in the third molar surgery, piezosurgery shows to be equivalent to conventional technique. The employment of one or another technique should be a personal decision of the surgeon.

Einleitung: Die Ausbildung eines Netzhautödems ist eine Hauptursache für die Verschlechterung des Sehvermögens bei ischämisch-hypoxischen und inflammatorischen Netzhauterkrankungen. Neben der erhöhten Permeabilität der Blut-Retina-Schranke trägt eine Wasserakkumulation in Netzhautzellen zur Ausbildung eines Netzhautödems bei. Müllerzellen regulieren die retinale Ionen- und Osmohomöostase, indem sie einen transzellulären Ionen- und Wassertransport vermitteln. Zudem kontrollieren Müllerzellen die Größe des Extrazellularraumes, indem sie bei neuronaler Aktivität eine Zellkörperschwellung – ausgelöst durch eine Verkleinerung der extrazellulären Osmolarität – verhindern. Unter pathologischen Bedingungen ist die Volumenregulation gestört, sodass Müllerzellen bei Hypoosmolarität anschwellen. Diese Müllerzellschwellung und eine Glutamat-induzierte Schwellung retinaler Neurone tragen zur Ausbildung eines zytotoxischen Netzhautödems bei. Neuroprotektive Faktoren wie BDNF (brain-derived neurotrophic factor) und bFGF (basic fibroblast growth factor) stimulieren das Überleben retinaler Neurone und verzögern so die retinale Degeneration. Zielstellung: Es war zu zu ermitteln, ob BDNF die zytotoxische Schwellung von Müller- und Bipolarzellen der Rattennetzhaut verhindert. Material und Methoden: Es wurden Netzhautschnitte und isolierte Müller- und Bipolarzellen von 55 adulten Long-Evans-Ratten (durchschnittlich 8-15 Zellen pro Versuchsreihe) verwendet. Eine osmotische Schwellung von Müller- und Bipolarzellen wurde durch eine Superfusion der Schnitte oder der Zellen mit einer 60%igen hypoosmolaren Lösung in Ab- oder Anwesenheit von Bariumchlorid induziert. Die maximale Querschnittsfläche von Müller- und Bipolarzellsomata wurde vor und nach einer vierminütigen Superfusion mit einem konfokalen Laserscanningmikroskop aufgezeichnet. Die nach der Superfusion ermittelte Querschnittsfläche wurde zu den anfänglich gemittelten Kontrollwerten in Beziehung gesetzt und prozentual als Mittelwert mit Standardfehler bestimmt. Mit Hilfe des Prism-Statistikprogramms (Graphpad) wurden die Ergebnisse mittels einem one-way ANOVA Test und einem nachfolgenden Bonferroni\'s multiple comparison Test sowie durch einen Mann-Whitney U Test statistisch analysiert. Ergebnisse: Bei Anwesenheit von BDNF wurde die osmotische Schwellung von Müllerzellen konzentrationsabhängig sowohl in Netzhautschnitten als auch in isolierten Zellen inhibiert. Ebenso inhibierte BDNF konzentrationsabhängig die Schwellung von Bipolarzellen in Netzhautschnitten, jedoch nicht in isolierten Zellen. In Schnitten von postischämischen Netzhäuten bewirkte BDNF eine Schwellungsinhibition von Müllerzellen, nicht aber von Bipolarzellen. Mit pharmakologischen Blockern wurde die durch BDNF induzierte Signalkaskade untersucht. Die BDNF-Schwellungsinhibition von Müllerzellen wurde durch eine Aktivierung von TrkB bewirkt. Die TrkB-Aktivierung führte in Müllerzellen zu einer Transaktivierung von FGF-Rezeptoren sowie zu einer Aktivierung einer glutamatergen-purinergen Signalkaskade, von der bekannt ist, dass sie die osmotische Müllerzellschwellung unterdrückt. Da bFGF die osmotische Müllerzellschwellung inhibiert, wird die Transaktivierung der FGF-Rezeptoren wahrscheinlich durch eine BDNF-induzierte Freisetzung von bFGF aus Müllerzellen vermittelt. Die Ergebnisse lassen vermuten, dass BDNF indirekt auf Bipolarzellen wirkt, indem es eine Freisetzung von Faktoren wie bFGF aus Müllerzellen induziert. Schlussfolgerungen: Die Schwellungsinhibition von Müller- und Bipolarzellen könnte ein neuroprotektiver Mechanismus von BDNF in der Netzhaut darstellen. Während BDNF direkt TrkB auf Müllerzellen aktiviert, ist die Inhibition der Bipolarzellschwellung indirekt und durch die Ausschüttung von glialen Faktoren wie bFGF vermittelt. Der Verlust des Effektes von BDNF auf die Bipolarzellschwellung in ischämischen Netzhäuten könnte darauf zurückzuführen sein, dass gliotische Müllerzellen keine glialen Faktoren mehr in Reaktion auf BDNF freisetzen. Der Verlust des glialen Einflusses auf die Bipolarzellvolumenhomöostase könnte zur Neurodegeneration in der ischämischen Netzhaut beitragen. / Introduction: Tissue edema is a major blinding complication of ischemic-hypoxic and inflammatory retinal diseases. In addition to the hyperpemeability of the blood-retinal barrier, water accumulation in retinal cells resulting in cellular swelling may contribute to the development of retinal edema. Müller glial cells regulate the retinal ion and water homeostasis by allowing transcellular ion and water fluxes. During neuronal activity Müller cells control the extracellular space volume by autocrine inhibition of cellular swelling caused by the reduction of extracellular osmolarity. However, under pathological conditions, Müller cells are not capable to regulate their volume so that they swell rapidly under hypoosmolarity. The osmotic swelling of Müller glial cells and the glutamate induced swelling of retinal neurons contribute to the development of cytotoxic retinal edema. Various neuroprotective factors including brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) stimulate the survival of retinal neurons and thus delay the retinal degeneration. Objective: The objective of the study is to determine whether BDNF inhibits the osmotic swelling of Müller and bipolar cells of the rat retina. Material and Methods: Retinal slices and freshly isolated Müller and bipolar cells of 55 adult Long-Evans rats (in average 8-15 cells per trial) were used. Osmotic swelling of Müller and bipolar cells was induced by superfusion of retinal slices or isolated cells with a 60% hypoosmotic extracellular solution in the absence or presence of barium chloride. The maximal cross-sectional area of Müller and bipolar cell somata was recorded before and after a four minute-long superfusion by using a laser scanning microscope. To determine the extent of cell soma swelling, the cross-sectional area of the cell body extent after superfusion was related to the former averaged cross-sectional area. Results were given as means with standard error as percent values. Statistical analysis was made with Prism (Graphpad) and the significance was determined by the One-way ANOVA test followed by Bonferroni\'s multiple comparison test and the Mann-Whitney U test, respectively. Results: We found that BDNF inhibits dose-depending the osmotic swelling of Müller cells in retinal slices and of isolated cells. BDNF also inhibited dose-depending the osmotic swelling of bipolar cells in retinal slices; however, it did not inhibit the osmotic swelling in isolated bipolar cells. In slices of postischemic retinas, BDNF inhibited the swelling of Müller cells but not the swelling of bipolar cells. The BDNF induced signal transduction cascade was examined by simultaneous administration of blocking agents with the receptor agonists in the hypoosmotic solution. The BDNF-induced inhibition of the osmotic Müller cell swelling was mediated by activation of TrkB. Activation of TrkB in Müller cells results in transactivation of FGF receptors and in an activation of a glutamatergic-purinergic signal transduction cascade which is known to inhibit the osmotic swelling of the cells. Since bFGF also inhibits the osmotic swelling of Müller cells, it can be assumed that the transactivation of FGF receptors is mediated by a BDNF-induced release of bFGF from Müller cells. The results suggest that the effect of BDNF on bipolar cells is indirect by inducing a subsequent release of glial factor from Müller cells such as bFGF. Conclusion: The results show that BDNF inhibits the osmotic swelling of Müller and bipolar cells. The inhibition of cytotoxic cell swelling may contribute to the neuroprotective action of BDNF in the retina. While BDNF acts directly in Müller cells, the BDNF-induced inhibition of the bipolar cell swelling is indirect and mediated by the release of glial factors such as bFGF from Müller cells. The abrogation of the BDNF-induced inhibition of the osmotic bipolar cell swelling in the postischemic retina could be explained with the impairment of the release of glial factors by Müller cells. The abrogation of the Müller cell-mediated regulation of the bipolar cell volume could contribute to the neuronal degeneration in the ischemic retina.

Descreve-se os achados epidemiológicos e clínico-patológicos das clostridioses diagnosticadas no Setor de Patologia Veterinaria da Universidade Federal do Rio Grande do Sul no período 1996-março/2010. Este estudo incluiu uma pesquisa retrospectiva nos arquivos do SPV e uma etapa prospectiva, a qual também teve o objetivo de desenvolver exames complementares específicos para cada clostridiose. As clostridioses mais prevalentes foram tétano (em equinos, bovinos, ovinos e caprinos), botulismo (em bovinos, suínos e aves) e enterotoxemia (em caprinos). Também houve casos de edema maligno em equinos, bem como de carbúnculo sintomático e hemoglobinúria em bovinos. Adicionalmente, foram coletadas amostras de soro sanguíneo de animais afetados por tétano, em diferentes estágios de evolução da doença, para subsequente inoculação em camundongos (testes de bioensaio) e comprovação da técnica no diagnóstico da enfermidade. / This study describes the epidemiological and clinicopathological findings of clostridial diseases diagnosed in the 1996-March, 2010 period in the Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul (SPV_UFRGS). A retrospective survey in the files of SPV was complemented with a prospective phase, which also aimed developing complementary diagnostic tests of clostridiosis. The most prevalent clostridiosis were tetanus (in horses, cattle, sheep and goats), botulism (in cattle, pigs and birds), and enterotoxemia in goats. There also were cases of malignant edema in horses, blackleg and bacillary hemoglobinuria in cattle. In addition, blood serum samples from animals affected by tetanus on different stages of the disease evolution were applied in mice bioassay, as a complementary diagnosing test for the disease.

Descreve-se os achados epidemiológicos e clínico-patológicos das clostridioses diagnosticadas no Setor de Patologia Veterinaria da Universidade Federal do Rio Grande do Sul no período 1996-março/2010. Este estudo incluiu uma pesquisa retrospectiva nos arquivos do SPV e uma etapa prospectiva, a qual também teve o objetivo de desenvolver exames complementares específicos para cada clostridiose. As clostridioses mais prevalentes foram tétano (em equinos, bovinos, ovinos e caprinos), botulismo (em bovinos, suínos e aves) e enterotoxemia (em caprinos). Também houve casos de edema maligno em equinos, bem como de carbúnculo sintomático e hemoglobinúria em bovinos. Adicionalmente, foram coletadas amostras de soro sanguíneo de animais afetados por tétano, em diferentes estágios de evolução da doença, para subsequente inoculação em camundongos (testes de bioensaio) e comprovação da técnica no diagnóstico da enfermidade. / This study describes the epidemiological and clinicopathological findings of clostridial diseases diagnosed in the 1996-March, 2010 period in the Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul (SPV_UFRGS). A retrospective survey in the files of SPV was complemented with a prospective phase, which also aimed developing complementary diagnostic tests of clostridiosis. The most prevalent clostridiosis were tetanus (in horses, cattle, sheep and goats), botulism (in cattle, pigs and birds), and enterotoxemia in goats. There also were cases of malignant edema in horses, blackleg and bacillary hemoglobinuria in cattle. In addition, blood serum samples from animals affected by tetanus on different stages of the disease evolution were applied in mice bioassay, as a complementary diagnosing test for the disease.