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[pt] O medo é uma parte intrínseca da evolução humana e animal, mas pode se manifestar de forma patológica, como no Transtorno de Ansiedade. Há estudos na literatura que apontam o uso modelos animais para compreender psicopatologias, devido a simplicidade e semelhança neurobiológica. Foram desenvolvidas duas linhagens de ratos, denominadas Cariocas com Alto e Baixo Congelamento (CAC e CBC), que significa os níveis de ansiedade. O objetivo deste estudo foi investigar comportamentos compulsivos e hiperativos nos Cariocas, especificamente o grooming e o rearing de 138 animais (CAC, n=34; CBC, n=36; CTL, n=38). A metodologia foi analítica e descritiva com base em imagens obtidas em gravações do teste do medo contextual, ao longo de 8 minutos antes do choque. Nossos resultados indicaram que os CBC apresentam maior frequência [F(3,60)= 45,94, p<0,0001] e duração [F(3,43)= 21,51, p< 0,0001] do comportamento de rearing quando comparados aos outros grupos, CAC e CTL (rANOVA; P<0,05, teste post hoc de LSD). Do mesmo modo, quanto ao grooming, houve uma diminuição significativa no tempo total [F(2,00)= 6,35, p<0,01]. Portanto, os resultados sugerem que os CBCs exibem um comportamento mais hiperativo, apoiando a hipótese de que podem servir como um modelo animal de TDAH. / [en] Fear is an intrinsic part of human and animal evolution, but it can manifest itself in pathological ways, such as Anxiety Disorder. There are studies in the literature that point to the use of animal models to understand psychopathologies, due to their simplicity and neurobiological similarity. Two strains of rats were developed, called Cariocas with High and Low Freezing (CAC and CBC), which means anxiety levels. The objective of this study was to investigate compulsive and hyperactive behaviors in Cariocas, specifically the grooming and rearing of 138 animals (CAC, n=34; CBC, n=36; CTL, n=38). The methodology was analytical and descriptive based on images obtained from recordings of the contextual fear test, over 8 minutes before the shock. Our results indicated that BCC present a higher frequency [F(3,60)= 45.94, p<0.0001] and duration [F(3,43)= 21.51, p< 0.0001] of behavior rearing when compared to the other groups, CAC and CTL (rANOVA; P<0.05, LSD post hoc test). Likewise, regarding grooming, there was a significant decrease in total time [F(2.00)= 6.35, p<0.01]. Therefore, the results suggest that CBCs exhibit more hyperactive behavior, supporting the hypothesis that they may serve as an animal model of ADHD.

Thesis (MScMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: Throughout history disease entities have been described which match the description of diseases now known to be caused by hantaviruses; however these viruses were first identified as the aetiologic agent in 1976, the first species named Hantaan virus after the river near which its natural host, the rodent species Apodemus agrarius, was captured. Since then numerous species in the Hantavirus genus, family Bunyaviridae, have been found, with today more than 30 species worldwide being known. Hantaviruses are hosted by rodents from the Muridae and Cricetidae families and by shrews (insectivores) in the Soricidae family. There are two types of hantavirus disease, Haemorrhagic fever with renal syndrome (HFRS) in the Old World and Hantavirus cardiopulmonary syndrome (HCPS) in the New World. The first two African hantaviruses were identified in 2006 in Guinea, West Africa; Sangassou virus (SANGV) in a rodent, the African wood mouse (Hylomyscus simus), and Tanganya virus (TGNV) in Therese’s shrew (Crocidura theresae). In this study, rodents and shrews were trapped at localities in the Western Cape and Northern Cape provinces of South Africa, and in the southern regions of Namibia. RNA was extracted from their lungs and screened for hantavirus sequences by RTPCR, using degenerate primers designed to detect all members of the Hantavirus genus. In addition, an in-house IgG ELISA assay was set up, based on recombinant N antigen from Dobrava virus, DOB-rN, and Puumala virus, PUU-rN. The assay was used to screen patient sera collected in an anonymous convenience serological survey using residual serum samples left over from routine testing at NHLS laboratories in the Western Cape for hantavirus-specific antibodies. RNA from 576 animal specimens was screened by RT-PCR; no hantavirus genome was detected in any of the specimens. Sera from 161 patients were screened for hantavirus antibodies; 11.18% of the sera were reactive to DOB-rN, 4.97% against PUU-rN and 2.48% against both antigens. v Though no virus was detected in the animals screened, this does not necessarily mean that there are no hantaviruses present in Southern Africa. A previous seroepidemiological survey conducted in South Africa reported on the presence of hantavirus specific antibodies by IFA in two species of rodents trapped in the Western Cape and Northern Cape Aethomys namquensis and Tatera leucogaster. Our was the second known study in South Africa conducted that determined and proved the presence of hantavirus specific antibodies in humans. / AFRIKAANSE OPSOMMING: Dwarsdeur die geskiedenis was daar beskrywings van siektes wat ooreenstem met die beskrywing van hantavirus simptome, maar die eerste etiologiese oorsaak van die siekte is eers in 1976 geïdentifiseer en Hantaan virus genoem, vernoem na die rivier waar naby die gasheer, Apodemus agrarius, gevang is. Van daar af het die soektog na nuwe hantavirusse intensief gevorder en vandag is daar meer as 30 spesies wêreldwyd wat aan die Hantavirus genus, ’n lid van die Bunyaviridae familie, behoort. Knaagdiere van die Muridae en Cricetidae families, sowel as spitsmuise (insekvreters) in die Soricidae familie is gasheer vir hantavirusse. Twee tipes hantavirus siekte is bekend, hemorragische koors met nier sindroom (HFRS) in die Ou Wêreld en hantavirus kardiopulmonale sindroom in die Nuwe Wêreld. Die eerste twee Afrika hantavirusse is in 2006 in Guinee Wes-Afrika geïdentifiseer; Sangassou virus (SANGV) in ’n knaagdier, die Afrika hout muis (Hylomyscus simus) en Tanganya virus (TGNV) in Therese se spitsmuis (Crocidura theresae). In hierdie studie is knaagdiere en spitsmuise op verskeie plekke in die Wes- en Noord-Kaap provinsies, asook die Suide van Namibië, gevang. RNS is onttrek vanuit die longe en hantavirus volgordes is gesoek deur middel RT-PKR deur gebruik te maak van Pan-Hanta primers wat ontwerp is om alle lede van die Hantavirus genus op te spoor. ’n Self-ontwerpde IgG ELISA, gebasseer op rekombinante N antigeen van Dobrava virus, DOB-rN en Puumala virus, PUU rN, is opgestel en gebruik om pasiënt serum, verkry in ’n anonieme serologiese opname, te toets; oorblywende serum, na toetse uitgevoer is deur NHLS laboratoriums in die Wes-Kaap, is verkry en getoets vir hantavirus spesifieke teenliggaampies. RNS van 576 dier monsters is getoets deur middel van RT-PKR en geen hantavirus is in enige van die monsters geïdentifiseer nie. Serum van 161 pasiënte is getoets vir hantavirus teenliggaampies; 11.18% van die serum was reaktief teen DOB-rN, 4.97% teen PUU-rN en 2.48% teen albei antigene. Alhoewel geen virus in die diere geïdentifiseer is nie, beteken dit nie noodwendig dat geen hantavirusse in Suidelike-Afrika voorkom nie. ‘n Vorige sero-epidemiologiese opname wat in Suid-Afrika gedoen is het die teenwoordigheid van hantavirus spesifieke teenliggaampies in twee knaagdier spesies, Aethomys namquensis en Tatera leucogaster gevang in die Wes-en Noord-Kaap, gevind. Ons studie is die tweede studie bekend in Suid-Afrika uitgevoer, wat die teenwoordigheid van hantavirus spesifieke teenliggaampies bevind en bewys het.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction: The anthracyclines (ACs), daunorubicin (DNR) and doxorubicin (DXR) are two of the most effective drugs known for the treatment of systemic neoplasms and solid tumours. However, their clinical use is often hampered by their dosedependent cumulative cardiotoxicity, which leads to irreversible and fatal druginduced congestive heart failure. The mechanism by which ACs induces heart damage is not fully understood. Recent reports have indicated that DXR activates autophagy and ubiquitin proteasome-mediated degradation of specific transcription factors, however, no reports exists on the effect of ACs on the E3 ubiquitin ligases, MuRF-1 and MAFbx. The aim of the first part of the study was therefore to investigate the effect of DNR treatment on the protein and organelle degradation systems in the heart and to elucidate the signalling mechanisms involved. Although this model was ideal in allowing the investigation of the signalling pathways which are affected by DNR, it did not allow for further exploration or manipulation of signalling pathways that may be of potential benefit in this context. The in vitro model was therefore used to validate the hypothesis that increased autophagy alleviates AC-induced cardiotoxicity and delays the onset of cardiomyocyte death. The aims for the second part of the study were (i) to characterize the effect of DXR in H9C2 cells, (ii) to determine whether the induction/inhibition of autophagy in combination with DXR alleviates cytotoxicity and (iii) to investigate the influence of increased/decreased autophagy in combination with DXR on reactive oxygen species (ROS) production, mitochondrial function, endoplasmic reticulum (ER) stress and the ubiquitin proteasome pathway. In the final part of this study, an in vivo model was used to assess the potential benefit of autophagy in a novel GFP-LC-3 tumour bearing mouse model of acute DXR-induced cardiotoxicity. Material and Methods: Adult rats were divided into two groups where one group received six intraperitoneal injections of 2 mg/kg DNR on alternate days and the other group received saline injections as control. Hearts were excised and perfused on a working heart system the day after the last injection and freeze clamped for biochemical analysis. H9C2s were cultured and treated with Bafilomycin A1 (10 nM, inhibitor of autophagy) for 6 hrs, Rapamycin (50 μM, inducer of autophagy) for 24 hrs, DXR (3 μM) for 24 hrs or a combination of these drugs. Following treatment, cells were harvested and assessed for cell death, proteolytic activity and oxidative stress using western blotting, fluorescence microscopy and flow cytometry. In the final phase of the study, twenty-four female mice were injected at 8 weeks with a mouse breast cancer cell line (EO771) and after observation of tumour growth, animals were either treated with one injection (i.p.) of Rapamycin (4 mg/kg), two injections (i.p.) of DXR (10 mg/kg) or a combination of the two drugs. After the experimental protocol, mice were terminated and their hearts were rapidly excised. The hearts were divided cross-sectionally and utilized for biochemical and histological analyses. Results and Discussion: DNR treatment significantly attenuated myocardial function and increased apoptosis in the ex vivo heart model. DNR-induced cardiac cytotoxicity was associated with the upregulation of two E3 ubiquitin ligases, MuRF-1 and MAFbx as well as a significant increase in two markers of autophagy, beclin-1 and LC-3. These changes observed in the heart were also associated with attenuation of the PI3-kinase/Akt signalling pathway. The augmentation of autophagy with rapamycin before DXR treatment significantly reduced cell death in the in vitro model. Indeed, rapamycin treatment demonstrated to be a vital survival mechanism for acute DXR-induced cardiotoxicity as it decreased cellular ROS production, improved mitochondrial function and prevented nuclear translocation of DXR. Moreover, these changes in cardiomyocytes were also associated with a reduction in the ubiquitin-proteasome pathway (UPP). In the final part of this study, a novel tumour bearing GFP-LC3 mouse model was developed to confirm the results obtained in the in vitro study. It was demonstrated that acute DXR-induced cardiotoxicity resulted in increased apoptosis, the inhibition of autophagy and increased proteolysis via the UPP. These findings were associated with a reduction in body weight and cardiomyocyte cross-sectional area. The cardiotoxic effects of DXR were substantially reduced when autophagy was induced with rapamycin. Taken together, our data strongly indicates that it is possible to attenuate the cardiotoxic effects of doxorubicin in cancer patients by carefully controlling the levels of autophagy using rapamycin as adjuvant therapy. / AFRIKAANSE OPSOMMING: Inleiding: Die antrasikliene (AC’s), daunorubisien (DNR) en doksorubisien (DKS), is twee van die mees effektiewe AC wat bekend is vir die behandeling van sistemiese neoplasmas en soliede tumore. Hulle kliniese gebruik word egter deur dosis afhanklike kumulatiewe kardiotoksisiteit benadeel, wat tot onomkeerbare en dodelike kongestiewe hartversaking kan lei. Die meganisme waardeur AC’s hartversaking kan veroorsaak, word nog nie ten volle verstaan nie. Onlangse navorsing het aangetoon dat DKS autofagie en die ubikwitienproteosoom-bemiddelde degradasie van spesifieke transkripsie faktore aktiveer. Daar is egter geen literatuur wat die effek van AC’s op die E3-ubikwitienligases, MuRF-1 en MAFbx beskryfnie. Die doel van hierdie eerste afdeling van die studie is om die effek van DNR behandeling op die proteïen- en organel degradasie sisteme in die hart te ondersoek en om van die betrokke seinmeganismes te bepaal. Alhoewel hierdie model ideaal is om sommige seinweë wat deur DNR geaffekteer word, te ondersoek, kon seinoordragpaaie wat potensieël voordelig in hierdie konteks is, nie in bg. model gemanipuleer word nie. Die in vitro model is gebruik om die hipotese dat verhoogde outofagie AC-geïnduseerde kardiotoksisiteit verlaag en sodoende seldood verminder, te bevestig. Die doel van hierdie afdeling van die studie was: (i) om die effek van DKS op H9C2 selle te karakteriseer, (ii) om te bepaal of die induksie/inhibisie van outofagie in kombinasie met DKS kardiotoksisiteit verbeter (iii) om die invloed van verhoogde/verlaagde outofagie in kombinasie met DKS op reaktiwe suurstof species (ROS), mitokondriale funksie, endoplasmiese retikulum (ER) stress en die ubikwitienproteosoompad te ondersoek. In die finale deel van hierdie studie, is ‘n in vivo model gebruik om die moontlike voordelige effek van verhoogde outofagie in ‘n GFP-LC-3 tumor-draende muismodel met akute DKSgeïnduseerde kardiotoksisiteit, ondersoek. Materiaal en Metodes: Volwasse rotte is in twee groepe verdeel waar een groep ses intraperitoneale inspuitings van 2 mg/kg DNR op afwissellende dae ontvang het en die andergroep as ‘n kontrole, ‘n soutoplossing gekry het. Die harte is verwyder en geperfuseer op ‘n werkende hartsisteem een dag na die laaste inspuiting en gevriesklamp vir biochemiese analises. H9C2 selle is vir 6 uurgekweek en behandel met Bafilomisien A1 (10 nM, ‘n autofagie inhibitor), 24 uur met Rapamisien (50 μM, ‘n autofagie induseerder), 24 uur met DKS (3 μM) of ‘n kombinasie van hierdie middels. Na behandeling is selle ge-oes vir analises in seldood, proteolitiese aktiwiteit en oksidatiewe stress deur van westelike kladtegniek, fluoresensie mikroskopie en vloeisitometrie gebruik te maak. In die finale fase van hierdie studie is vier en twintig, agt weke oue wyfie muise ingespuit met ‘n muisborskankersellyn (E0771) en is tumorgroei waargeneem; die diere is of behandel met een rapamisien inspuiting (i.p) (4 mg/kg), of twee DKS inspuitings (i.p.) (10 mg/kg) of ‘n kombinasie van die twee middels. Na die eksperimentele protokol, is die muise van kant gemaak en hulle harte vinnig verwyder. Die harte is in twee verdeel en gebruik vir biochemiese- en histologiese analises. Resultate en Bespreking: DNR behandeling het kardiale funksie betekenisvol verswak en apoptose in die hart verhoog. DNR-geïnduseerde kardiotoksisiteit is geassosieer met die opregulering van E3-ligases, MuRF-1 en MAFbx en het ook ‘n betekenisvolle toename in twee outofagie merkers, beclin-1 en LC-3 veroorsaak. Hierdie veranderinge wat in die hart waargeneem is, is ook geassosieer met ‘n onderdrukking van die PI3-kinase/Akt seinweg. Die toename in outofagie met rapamisien voor DKS behandeling het seldood in die vorm van apoptose betekenisvol verlaag. Daarmee saam het verhoogde outofagie ‘n noodsaaklike oorlewings meganisme vir akute DKS-geïnduseerde kardiotoksisiteit gedemonstreer. Die rede hiervoor is dat dit ROS produksie verlaag het, mitokondriale funksie verbeter het en DKS translokasie vanuit die sitoplasma tot binne die nukleus verhoed het. Hierdie veranderinge in kardiomiosiete is ook met ‘n afname in die ubikwitienproteosoomseinweg (EPS) geassosieer. In die finale deel van hierdie studie, is ‘n nuwe tumor-draende muismodel ontwikkel om die resultate wat in die in vitro studie gekry is, te bevestig. Daar is bewys dat akute DKS-geïnduseerde kardiomiotoksisiteit aanleiding gegee het tot verhoogde apoptose, outofagie inhibisie en verhoogde proteolise via die EPS. Hierdie bevindinge is geassosieer met ‘n verlaging in liggaamsgewig en kardiomiosiet dwarssnit area. Die kardiotoksiese effekte van DKS is insiggewend verminder as autofagiege ïnduseer is met rapamisien. Om saam te vat: Ons data bevestig dat dit moontlik is om die kardiotoksiese effekte van DKS in kanker pasiënte te verminder deur outofagie vlakke te monitor en te kontroleer deur middel van rapamisien behandeling as bykomende terapie.

Le cannabis produit de nombreux effets psychologiques et physiologiques sur le corps humain. Les molécules contenues dans cette plante, désignées comme « phytocannabinoïdes », activent un système endogène qu’on appelle le système endocannabinoïde (eCB). Les effets de la consommation de cannabis sur la vision ont déjà été décrits sans cependant de formulation sur les mécanismes sous-jacents. Ces résultats comportementaux suggèrent, malgré tout, la présence de ce système eCB dans le système visuel, et particulièrement dans la rétine. Cette thèse vise donc à caractériser l’expression, la localisation et le rôle du système eCB dans la rétine du singe vervet, une espèce animale ayant un système visuel semblable à celui de l’humain. Nous avons mis au point un protocole expérimental d’immunohistochimie décrit dans l’article apparaissant dans l’Annexe I que nous avons utilisé pour répondre à notre objectif principal. Dans une première série de quatre articles, nous avons ainsi caractérisé l’expression et la localisation de deux récepteurs eCBs reconnus, les récepteurs cannabinoïdes de type 1 (CB1R) et de type 2 (CB2R), et d’un 3e présumé récepteur aux cannabinoïdes, le récepteur GPR55. Dans l’article 1, nous avons démontré que CB1R et une enzyme clé de ce système, la fatty acid amide hydrolase (FAAH), sont exprimés dans les parties centrale et périphérique de la rétine, et abondamment présents dans la fovéa, une région où l’acuité visuelle est maximale. Dans l’article 2, nous avons localisé le CB2R dans des cellules gliales de la rétine : les cellules de Müller et nous avons proposé un modèle sur l’action de cette protéine dans la fonction rétinienne faisant appel à une cascade chimique impliquant les canaux potassiques. Dans l’article 3, nous avons observé le GPR55 exclusivement dans les bâtonnets qui sont responsables de la vision scotopique et nous avons soumis un deuxième modèle de fonctionnement de ce récepteur par le biais d'une modulation des canaux calciques et sodiques des bâtonnets. Vu que ces 3 récepteurs se retrouvent dans des cellules distinctes, nous avons suggéré leur rôle primordial dans l’analyse de l’information visuelle au niveau rétinien. Dans l’article 4, nous avons effectué une analyse comparative de l’expression du système eCB dans la rétine de souris, de toupayes (petits mammifères insectivores qui sont sont considérés comme l’étape intermédiaire entre les rongeurs et les primates) et de deux espèces de singe (le vervet et le rhésus). Ces résultats nous ont menés à présenter une hypothèse évolutionniste quant à l’apparition et à la fonction précise de ces récepteurs. Dans les articles subséquents, nous avons confirmé notre hypothèse sur le rôle spécifique de ces trois récepteurs par l’utilisation de l’électrorétinographie (ERG) après injection intravitréenne d’agonistes et d’antagonistes de ces récepteurs. Nous avons conclu sur leur influence indéniable dans le processus visuel rétinien chez le primate. Dans l’article 5, nous avons établi le protocole d’enregistrement ERG normalisé sur le singe vervet, et nous avons produit un atlas d’ondes ERG spécifique à cette espèce, selon les règles de l’International Society for Clinical Electrophysiology of Vision (ISCEV). Les patrons électrorétinographiques se sont avérés semblables à ceux de l’humain et ont confirmé la similarité entre ces deux espèces. Dans l’article 6, nous avons démontré que le blocage de CB1R ou CB2R entraine une modification de l’électrorétinogramme, tant au niveau photopique que scotopique, ce qui supporte l’implication de ces récepteurs dans la modulation des ondes de l’ERG. Finalement, dans l’article 7, nous avons confirmé le modèle neurochimique proposé dans l’article 3 pour expliquer le rôle fonctionnel de GPR55, en montrant que l’activation ou le blocage de ce récepteur, respectivement par un agoniste (lysophosphatidylglucoside, LPG) ou un antagoniste (CID16020046), entraine soit une augmentation ou une baisse significative de l’ERG scotopique seulement. Ces données, prises ensemble, démontrent que les récepteurs CB1R, CB2R et GPR55 sont exprimés dans des types cellulaires bien distincts de la rétine du singe et ont chacun un rôle spécifique. L’importance de notre travail se manifeste aussi par des applications cliniques en permettant le développement de cibles pharmacologiques potentielles dans le traitement des maladies de la rétine. / Cannabis produces a range of psychological and physiological effects on the human body. Cannabinoids are the chemical compounds found in cannabis that activate an endogenous system, termed the endocannabinoid (eCB) system. Reports made in the 1970s have noted that cannabis consumption affects vision. It is therefore suggested that the eCB system is present in the visual system, particularly in the retina. This thesis aims at characterizing the expression, localization, and role of the eCB system in the vervet monkey retina. This animal model has a similar visual system as humans. Using immunohistochemistry methods presented in the article of Annexe I, we have established an experimental protocol to answer our goal. In the first series of four articles, we have characterized the expression and localization of the cannabinoid receptor 1 (CB1R), cannabinoid receptor 2 (CB2R), and the putative cannabinoid receptor GPR55. In Article 1, we have demonstrated that CB1R and a key enzyme of this system, FAAH (fatty acid amide hydrolase), are expressed in the central and peripheral retina, but heavily present in the fovea, the retinal region responsible for high acuity vision. In Article 2, we have localized CB2R in the glial Müller cells and hypothesized a possible mechanism of action of CB2R involving potassium buffering. In Article 3, we found that GPR55 is exclusively expressed in rods and have proposed its role through the modulation of calcium and sodium channels in rods. Given that these three receptors are segregated in the vervet monkey retina, we suggested that they might have distinct roles in retinal physiology. In Article 4, we reported a comparative analysis of the expression of the eCB system components in the retina of rodents, tree shrews (small mammals considered as early primates), and monkeys. This paper provides evidence that the eCB system is differently expressed in the retina of these mammals and suggests a distinctive role of eCBs in visual processing. In the subsequent series of three articles, we confirmed their suggested roles in the retina by using electroretinography (ERG) and intravitreal injections of agonist and antagonist of these receptors. We concluded that they indeed play important roles in the retina. In Article 5, we developed a standard protocol for ERG testing in our animal model and have published an ERG atlas with normalized amplitudes and latency values similar to that of humans, following the guidelines of the International Society for Clinical Electrophysiology of Vision. In Article 6, we showed that blockade of CB1R or CB2R with specific antagonists modifies the ERG, both in photopic and scotopic conditions, which confirms the implication of these receptors in normal retinal function. Finally, in Article 7 (expression of GPR55 in rods only), we confirmed the suggest role of GPR55 in rods by showing that activation or blockade of GPR55 with a specific agonist (lysophosphatidylglucoside) or antagonist (CID16020046) increases or decreases the amplitude of the scotopic ERG waveforms. Taken together, these articles demonstrate that CB1R, CB2R, and GPR55 are differentially expressed in the vervet monkey retina and have distinct roles. This work has also clinical relevance in the way that we have discovered new pharmacological targets that can be used for treatment of many retinal diseases.

Les rongeurs commensaux sont responsables de grands dommages en agriculture et dans les zones urbaines. En tant qu’espèces invasives, elles peuvent mettre en danger les espèces locales et sont porteurs et vecteurs de plusieurs zoonoses importantes. Les méthodes de contrôle sont basées principalement sur l’utilisation des warfarines, lesquelles produisent un grand nombre d’intoxications sur des espèces non ciblées et ont perdu une partie de leur efficacité à cause des résistances génétiques constatées chez les espèces cibles. De plus, ces méthodes sont considérées comme inhumaines parce qu’elles causent une mort lente et douloureuse par hémorragies. L’olfaction est une source principale d’évaluation des risques présents dans l’environnement pour les rongeurs, avec la perception des signaux chimiques des prédateurs ou signaux de toxicité des plants/nourriture. Cette perception olfactive peut être utilisé pour modifier l’utilisation de l’espace des rongeurs. L’objectif de cette thèse était l’identification des réponses comportementales aux messages chimiques importants (par exemple les signaux chimiques émis par les plantes et les prédateurs) dans l’écologie de la souris domestique (Mus musculus), avec l’utilisation de souches de laboratoire comme modèle des animaux sauvages. Nos résultats ont montré que la souris a évité de façon significative les signaux chimiques complexes du furet et un signal chimique ubiquitaire des plantes, lié à la maturation et la pourriture des aliments (l’éthanol). La protéine du chat Fel d 1, laquelle fait partie de la famille des sécrotoglobines et est un allergène majeur du chat, n’a pas modifié le comportement d’exploration de la souris ou son comportement de recherche et de consommation de nourriture. Le composant chimique des fèces de renard, le TMT a induit un évitement clair et des réponses de stress comme cela a été rapporté dans la littérature. De plus, j’ai fait une revue de la littérature pour évaluer et discuter les méthodes de contrôle des rongeurs d’un point de vue éthique, revue qui a démontré que les méthodes actuelles peuvent être considérés inhumaines et ne correspondent pas aux attentes actuelles de la société et aux standards sur le bien-être dans d’autres domaines comme les élevages de production ou les animaux de laboratoire. Ces résultats ouvrent des nouvelles voies de recherche afin d’identifier les composants chimiques du furet et des plantes liés au comportement d’évitement des rongeurs, les prochaines étapes utilisant des animaux sauvages à la fois en laboratoire et sur le terrain. / Rodent commensal species produce great damage in agriculture and urban areas. As invasive species they can endanger local species and are carriers and vectors of several important zoonoses. Control methods rely mainly on the use of warfarins, which can be inadvertently be taken up by untargeted species. Warfarins have also lost their efficacy in rodents due to the development of genetic resistance. In addition, these methods are considered inhumane as they cause a slow and painful death due to haemorrhages. Olfaction is a main source for environmental risk assessment by rodents, and it can be used to modify their use of space. My aim in this thesis was to identify behavioural reactions of the house mouse (Mus musculus), using laboratory strains as models of wild animals, to ecologically meaningful chemical messages, including predator and plant chemical olfactory cues. My results showed that mice avoided complex ferret olfactory cues and ethanol which is a ubiquitous chemical related to fruit rotting and ripening. The feline protein Fel d 1, which belongs to the secretoglobin family and is a major cat allergen in humans, did not elicit significant avoidance or alter foraging behaviour in mice. However, Trimethylthiazoline purified from fox faeces, elicited clear avoidance behaviour and stress responses. I carried out a bibliographic review to evaluate and discuss rodent pest control methods from an ethical standpoint. This literature showed that many of the current methods of pest control are considered inhumane, and do not tally with current society concerns and welfare standards in other domains such as farms or laboratory animals. These results raise new research questions to identify ferret and plant chemical compounds that can induce rodent avoidance, and to carry out next stage of research with wild animals both under laboratory and field conditions.

O ouriço-preto (Chaetomys subspinosus, Olfers 1818), roedor endêmico da Mata Atlântica ameaçado de extinção, foi estudado na região cacaueira do sul da Bahia. O conhecimento acerca de aspectos básicos de sua história natural ainda é insipiente. Neste contexto, foi estudada a ecologia comportamental do ouriço-preto quanto à ecologia alimentar, organização temporal do comportamento, uso do espaço e seleção do habitat, a fim de conhecer suas estratégias adaptativas, seus requerimentos de habitat e embasar ações de conservação. Foram rádio-monitorados quatro indivíduos em vida-livre, por 11 a 13 meses cada, durante 24 dias completos (24h) e 146 noites parciais, totalizando 1.520 horas de observação, entre abril de 2005 a setembro de 2006. Dados sobre o comportamento, localização e uso de recursos foram registrados a cada 10 minutos por visualização direta dos animais. Simultaneamente, foram coletados dados sobre: clima, disponibilidade de alimento, fase da lua, tipo de vegetação na paisagem, estrutura horizontal e vertical da vegetação, abundância de recursos, e composição nutricional de espécies da dieta. Os dados foram analisados através de três abordagens. Os resultados do primeiro enfoque revelam que os ouriços-preto estudados foram estritamente folívoros e especialistas, e não apresentaram mudanças na composição da dieta ao longo do ano. Preferiram folhas jovens, espécies pioneiras, principalmente Fabaceas com capacidade de fixação biológica de nitrogêncio. Os resultados do segundo enfoque indicaram que os ouriços estudados são estritamente noturnos, solitários, possuem baixos níveis de atividade entre outras estratégias voltadas para a conservação de energia. Eles apresentaram ritmo circadiano de atividade e padrão bimodal de atividade que parecem associados a aspectos que permeiam o processo digestivo e de forrageamento. Não mostraram expressivas mudanças comportamentais ao longo do ano, mas em noites chuvosas sua atividade foi reduzida. Os resultados da terceira abordagem indicam que eles possuem áreas de vida pequena, são estritamente arborícolas, e preferem locais com alta complexidade vertical da vegetação e infestação de cipós em todas as escalas de seleção de habitat avaliadas. Dessa forma, as florestas e as áreas de borda foram preferidas pelos ouriçospreto, e raramente foram usadas as capoeiras e sistemas agroflorestais. Este estudo comprova que os ouriços-preto são estritamente folívoros, arborícolas e especialistas. Eles adotam estratégias comportamentais voltadas à conservação de energia, similares a outros mamíferos folívoros, e são altamente seletivos no uso de recursos do habitat. Estes recursos são comuns em áreas florestais sobre perturbação do efeito de borda. Enquanto, por um lado os ouriços-preto mostram ser especializados em bordas de floresta, por outro, evitam áreas forestais estruturalmente simplificadas. A tolerância ao efeito de borda e a evitação dos sistemas agroflorestais pelos ouriços-preto são discutidas no âmbito de sua conservação. / The behavioral ecology of thin-spined porcupine (Chaetomys subspinosus, Olfers 1818), threatened rodent endemic from Atlantic forest, was researched in the cacao-growing region of southern Bahia. The knowledge about basic aspects of specie natural history is still incipient. The aim of the present research was to study feeding ecology, behavior temporal organization, use of space, and habitat selection, to know the adaptation strategies, their habitat requirements and to support conservation actions. In this way, four individuals in situ were followed using radiotelemetry along 11 to 13 months each, during 146 half-nights and 24 complete days (24h), totaling 1520 hours of observation, from April 2005 to September 2006. Data about behavior, localization and use of resources were registered by instantaneous sampling performed each 10 minutes by direct visualization of animals. Simultaneously, data about climatic variables, food availability, moon phase, vegetation types in landscape, horizontal and vertical vegetation structure, resource abundance, and nutritional composition of diets species were collected. Data was analyzed following three different approaches. The results of the first part revealed that the thin-spined porcupines are strictly folivorous and specialist, eating a lot of few species, any change on foraging patterns and diet composition were observed along the year. The preference was for young leaves, pioneer species, mainly Fabaceas with capability of biological nitrogen fixation. The results of the second part indicated that the studied thin-spined porcupines are strictly nocturnal and solitary presenting low level of activity among other strategies for energy conservation. Also, presented circadian rhythm and activity bimodal pattern related to aspects that permeate their foraging and digestive process. They did not change their activities or dislocations due to seasonal but in raining nights their activity was reduced. The results of the third approach revealed that they exhibit small home range, were strictly arboreal and preferred locals with high complexity vertical vegetation and lianas abundance in all the habitat-scales selection evaluated. Thus the forest and the edged areas were preferred by the porcupine, and rarely went to early-growth forest and agro-forest systems. This study confirmed that thin-spined porcupine is strictly folivore, arboreal and specialist. They adopt conservative-energy strategies similar to others arboreal folivorous mammals, and denotes that are highly selective in use habitat resources. Those resources are common in forest areas with edge effect disturbance. While demonstrated to be specialist in edges forest, in other side, they avoid simplified structurally areas. The thin-spined porcupine tolerance to edge effect and their avoidance to agroforest systems are discussed under the focus of conservation.

O gânglio estrelado (GE) é o principal componente da inervação cardíaca extrínseca e está envolvido na gênese de diversas cardiomiopatias. Durante o envelhecimento, o controle neural do coração dos mamíferos é alterado de forma complexa e não clara, geralmente ocasionando decremento da função cardíaca e maior propensão a doenças degenerativas. A ocorrência de resultados dissonantes quanto aos parâmetros morfoquantitativos durante o envelhecimento, como o aumento ou diminuição do número total de neurônios simpáticos, é assunto para discussões interessantes. Esta pesquisa foi conduzida em preás machos (Galea spixii), um pequeno roedor da fauna brasileira. Desta forma, estudou-se o efeito do desenvolvimento pós-natal (maturação e envelhecimento) na macro e microestrutura do gânglio estrelado esquerdo (GEe) de preás, por meio de microscopia quantitativa tridimensional (Estereologia) associada a técnicas de imuno-histoquímica. De acordo com a fase específica do desenvolvimento pós-natal, os animais foram alocados nos seguintes grupos etários: Neonatos, Jovens, Adultos e Senis. Inicialmente, os animais foram submetidos à eutanásia e seus gânglios estrelados esquerdos coletados e fixados em solução de formaldeído (4%) em PBS. Foi realizada amostragem sistemática e uniformemente aleatória (SURS), estimando-se: volume do GEe, volume neuronal e número total de neurônios do GEe. Os principais achados deste estudo foram: i) aumento do comprimento do gânglio - 42% entre Neonato e Senil; 34% entre Jovem e Senil e 35% entre Adulto e Senil; ii) hipertrofia do GEe - 171% entre os grupos Neonato e Adulto; iii) aumento do tecido não neuronal - 268% entre os grupos Neonato e Adulto; iv) estabilidade no número total de neurônios uninucleados, binucleados e total (uni+bi); v) estabilidade no tamanho (volume) dos neurônios uninucleados e binucleados; e vi) estabilidade no número total de neurônios imunorreativos ao Ki-67 (uni+bi). Espera-se que os resultados gerados por esta pesquisa possam esclarecer alguns aspectos estruturais da plasticidade neural durante o desenvolvimento pós-natal de preás, avançando assim o conhecimento acerca da inervação cardíaca extrínseca / The stellate ganglion (SG) is a main component of extrinsic cardiac innervation and is involved in the genesis of various cardiomyopathies. During ageing, the neural control of heart in mammals is altered in the complex shape and unclear, generally cause decrement in the cardiac function and a greater propensity to degenerative diseases. The occurrence of discordant results regarding the morphoquantitative parameters during ageing, such as increase or decrease in the total number of sympathetic neurons, is a subject for interesting discussions. This research was conducted in males preas (Galea spixii), a small rodent of the Brazilian fauna. Therefore, this work aimed to study the effect of postnatal development (maturation and ageing) in the macro and microstructure of the left stellate ganglion (LSG) in preas by dimensional quantitative microscopy (Stereology) associated to immunohistochemistry techniques. According to a specific stage of postnatal development, the animals were allocated into the following age groups: Newborn, Young, Adult and Elderly. The animals were euthanised and the left stellate ganglia were collected and fixed in 4% formaldehyde solution in PBS. A systematic uniformly random sampling (SURS) was performed to estimate: the volume of LSG, neuron volume and the total number of LSG neurons. The main findings of this study were: i) increase in length ganglia - 42% between Newborn and Elderly; 34% between Young and Elderly and 35% between Adult and Elderly; ii) hypertrophy of LSG - 171% between the groups Newborn and Adult; iii) increase of non-neuronal tissue - 268% between the groups Newborn and Adult; iv) stability for the total number of uninucleate neurons, binucleate neurons and total (uni+bi); v) stability in the size (volume) of uninucleate and binucleate neurons; and, vi) stability for the total number of neurons immunorreactive to Ki-67 (uni+bi). It is expected that the results generated for this research may clarify structural aspects of neural plasticity during the postnatal development of preas, thus advancing the knowledge about the extrinsic cardiac innervation

O conhecimento sobre hantavirus patogênicos no Brasil até o momento indica que roedores silvestres são os reservatórios naturais. Estes roedores podem eliminar grandes quantidades do vírus através das fezes, urina e saliva (Khaiboullina et al., 2005). A infecção humana pode ocorrer por contato com roedores ou inalação dos aerossóis que contêm o vírus. A síndrome cardiopulmonar por hantavirus (HCPS) tem sido relatada em humanos em muitas regiões do Brasil. Tendo em vista a quantidade de casos de doenças emergentes e re-emergentes nas últimas décadas elegemos algumas regiões para um estudo detalhado sobre a distribuição desses reservatórios virais e consequentemente a caracterização da variante circulante. Com objetivo de adquirir um diagnóstico rápido e sensível, desenvolvemos primers específicos que pela técnica de Real- time RT- PCR é capaz de detectar o genoma viral em apenas uma hora em amostras de pulmões, rins e urina de roedores. Ao todo, nosso trabalho analisou 153 amostras sendo: 126 de roedores silvestres e domésticos, (de cinco diferentes regiões do Estado de São Paulo onde: 10 amostras colhidas no município de Jacupiranga, 61 oriundas de Teodoro Sampaio, 7 de Salesópolis, 48 provenientes de Biritiba Mirim, 16 de Nazaré Paulista) e mais 27 soros de pacientes de diferentes localidades de Minas Gerais. Neste estudo foi possível detectar dez amostras positivas em Biritiba Mirim, quatro em Nazaré Paulista e em vinte e dois soros de pacientes com hantavirose. O monitoramento contínuo através de testes moleculares em animais silvestres é imprescindível para um melhor entendimento da circulação dos hantavirus nessas regiões. Acreditamos que este teste possa auxiliar na vigilância e também no diagnóstico dos hantavirus no Brasil. / Current knowledge of the pathogenic hantavirus indicates that wild rodents are its primary natural reservoir. These rodents can eliminate large amounts of the virus through feces, urine and saliva. Human infection can occur through contact with rodents or inhalation of aerosols containing of the virus. Hantavirus cardiopulmonary syndrome (HCPS) has been reported in humans in many regions of Brazil. In view the number of cases of emerging diseases and re- emerging in recent decades, some regions to were select a detailed study on the distribution of these viral reservoirs, and consequently viral characterization. We developed specific primers to detect the presence of viral genomes using Real- time RT- PCR (Reverse- Transcriptase Polymerase Chain Raction). Altogether, our study analyzed 153 samples: 126 of wild rodents and domestic, of five different regions of São Paulo. 10 samples collected in Jacupiranga municipality, 61 in Teodoro Sampaio, 7 Salesópolis, 48 samples in Biritiba Mirim, 16 of Nazaré Paulista and 27 samples of the serum from patients from different sites of Minas Gerais State. Their samples were tested for hantavirus using the described SYBR Green-based real-time RT-PCR protocol and the specific primers. The presence of hantavirus RNA was detected in 10 rodents from Biritiba Mirim, 4 in Nazaré Paulista and 22 in serum of patients with hantaviruse. The surveillance by molecular tests in wild animals is essential to understood of hantavirus circulation in these regions. This SYBR Green real-time RTPCR method for detection of hantavirus may be useful for surveying hantaviruses in Brazil.

Submitted by Flávio Nunes (fnunes) on 2015-03-13T18:58:18Z No. of bitstreams: 1 MaurySayaoLobatoAbreu.pdf: 4129833 bytes, checksum: 497b7547567dfc77dbc9b7f62f812649 (MD5) / Made available in DSpace on 2015-03-13T18:58:18Z (GMT). No. of bitstreams: 1 MaurySayaoLobatoAbreu.pdf: 4129833 bytes, checksum: 497b7547567dfc77dbc9b7f62f812649 (MD5) Previous issue date: 2012 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste estudo investiguei os padrões de uso do espaço arbóreo e terrestre por pequenos mamíferos não-voadores em diferentes estratos florestais em uma área de floresta com araucária (domínio da Mata Atlântica) localizada em São Francisco de Paula sul do Brasil. Os espécimes foram capturados, marcados e recapturados em quatro amostragens, cada uma com seis noites de duração, entre fevereiro e novembro de 2011. As armadilhas foram instaladas no solo, no sub-bosque e no dossel (50 em cada estrato) em uma grade de 10x10 estações de captura. Com base na estrutura da vegetação, determinei a complexidade e heterogeneidade do habitat em cada estação de captura. Através de métodos coleta e contagem, estimei a disponibilidade de recursos alimentares (frutos, sementes e invertebrados) para cada estrato e medi 12 variáveis de microhabitat ligadas à estrutura da vegetação. Procurei verificar a existência de associação entre complexidade e heterogeneidade com a diversidade e riqueza de pequenos mamíferos. Além disso, testei a hipótese de associação entre as espécies e os estratos, o uso dos estratos e a disponibilidade de recursos alimentares e entre as variáveis de microhabitat e a abundância das espécies. Como resultados, encontrei um total de oito espécies de roedores e duas de marsupiais durante todo o estudo, com nove espécies presentes no solo, sete no sub-bosque e quatro no dossel. Três espécies foram essencialmente terrestres (Akodon serrensis, Thoptomys nigrita e Monodelphis dimidiata) e uma foi essencialmente arborícola (Juliomys sp.). Não houve associação entre complexidade e heterogeneidade com a riqueza e diversidade de pequenos mamíferos (P > 0,1 para todas as comparações). Contudo, detectei que as espécies mais abundantes apresentaram preferência por um dos estratos, sendo A. montensis, A. serrensis e Delomys dorsalis altamente associadas ao solo (P < 0,005). O marsupial Gracilinanus microtarsus foi a única espécie de pequeno mamífero associada significativamente à disponibilidade de recurso alimentar no dossel (P < 0,01). A maioria dos pequenos mamíferos foi influenciada por variáveis de microhabitat diferentes. A ausência de correlação da complexidade e da heterogeneidade do habitat com a riqueza e diversidade dos pequenos mamíferos possivelmente está associada à escala espacial e escala temporal que utilizei. Os resultados sugerem que a frequência de uso dos estratos pelos pequenos mamíferos não é fortemente influenciado pela disponibilidade de alimento, mas possivelmente por outros fatores como predação e/ou competição. As preferências por microhabitat pelas espécies são condizentes com as preferências por estratos verticais, e explicaram grande parte da ocupação do espaço pelos pequenos mamíferos. / In this study I investigated patterns in the use of arboreal and terrestrial space by non-volant small mammals on different forest layers in an araucaria forest area (Atlantic Forest domain) in São Francisco de Paula municipality Southern Brazil. The specimens were captured, marked and recaptured during four sampling periods, each one with six nights of sampling, between February and November 2011. The traps were installed on the ground, understory and canopy (50 on each) on a 10x10 grid. The habitat complexity and heterogeneity was determined in each trap stations based on vegetation structure. I estimated the food resource availability (fruits, seeds and invertebrates) for every layer using collect and counting methods, and I also measured 12 microhabitat variables related to vegetation structure. In order to verify association of the complexity and heterogeneity with the diversity and richness of small mammals I used the Pearson linear correlation test. Furthermore, I tested the association hypothesis between species and layers, layer use and food resource availability, and the microhabitat variables and the species abundance. As results, I found eight rodent species and two marsupials during the whole study, with nine species on the ground, seven on the understory, and four on the canopy. Three species was essentially terrestrial (Akodon serrensis, Thaptomys nigrita and Monodelphis dimidiata) and one was essentially arboreal (Juliomys sp.). There was no association of the complexity and heterogeneity to the small mammals richness and diversity (P > 0,1 for all comparisons). However, I detected that the most abundant species showed a preference for some layer, with Akodon montensis, A. serrensis and Delomys dorsalis highly associated with the ground (P < 0,005). The marsupial Gracilinanus microtarsus was the only small mammal species significantly associated to the resource availability on the canopy (P < 0,01). The majority of small mammals was influenced by different microhabitat variables. The absence of correlation of the habitat complexity and heterogeneity with the small mammals’ richness and diversity was possibly due to the spatial and temporal scale used. The results suggested that the frequency of use of layers by small mammals were not strongly influenced by food availability, but probably by other factors like predation risk and competition. The microhabitat preferences of the species were consistent with the vertical layer used, and explained a great part of the space use by small mammals.