Skin is the largest immune organ and an ideal site to administer vaccines. However, by nature, skin is not permeable to antigens, which are macromolecules. The major hurdle in skin permeation is the outermost stratum corneum layer. Microneedles have proven feasible to create micron-sized channels in the epidermis of the skin, through which protein and plasmid DNA antigens can penetrate into the viable skin epidermis and dermis. However, the immune responses induced by microneedle-mediated transcutaneous immunization with protein or plasmid DNA alone are generally weak, and a vaccine adjuvant is often required to induce strong immune responses. Data from numerous previous studies have shown that nanoparticles as a vaccine carrier can significantly enhance the immunogenicity of antigens, but the feasibility of utilizing nanoparticles as a vaccine carrier to enhance the immune responses induced by microneedle-mediated transcutaneous immunization has rarely been studied. In this dissertation, using protein antigen (OVA) chemically conjugated onto the surface of solid-lipid nanoparticles and plasmid DNA (pCMV-beta, pVax/opt-BoNT/C-Hc50, and pCI-neo-sOVA) physically coated on the surface of cationic polymeric nanoparticles, we showed that the immune responses induced by microneedle-mediated transcutaneous immunization with protein antigens or plasmid DNA vaccines are significantly enhanced by delivering the proteins and plasmid DNA with nanoparticles. Importantly, microneedle-mediated transcutaneous immunization with proteins or plasmid DNA induces not only systemic immune responses, but also mucosal immune responses. In addition, it is generally believed that microneedles are safe. However, it remained unclear whether the micropores created by microneedles on the skin will also facilitate the permeation of microbes such as bacteria into the skin. In this dissertation, we also designed an unique ex vivo model to evaluate the permeation of live bacteria through mouse skin pretreated with microneedles. The results demonstrated that the risk of potential bacterial infection associated with microneedle treatment is not greater than that associated with a hypodermic needle injection. / text

β-N-methylamino-L-alanine (BMAA) is one of the more recently discovered bioactive compounds produced by cyanobacteria. BMAA is a non-protein amino acid reported present in human brain tissues of patients deceased from a neurodegenerative disease, such as Alzheimer´s disease or amyotrophic lateral sclerosis (ALS). This observation in combination with its neurotoxic effects in eukaryotes (in vivo and in vitro) and its potential to incorporate into (human) proteins, causing protein aggregation, suggests BMAA as a possible causative environmental agent for neurodegenerative diseases. Due to the ubiquitous nature of cyanobacteria with a wide occurrence in both aquatic and terrestrial environments, BMAA could be globally spread. Hence, investigations of a possible coupling between BMAA and neurodegeneration are urgently needed as well as to identify sources of BMAA in Nature. The aim of this thesis was to examine the potential occurrence of BMAA in bloom forming cyanobacteria of the Baltic Sea and its possible transfer to other organisms of this ecosystem. Of importance was also to reveal any likely routes for human BMAA exposure in the Baltic Sea region and to further investigate BMAA as a triggering agent for neurodegenerative diseases. Acknowledged difficulties of analysing BMAA in biological samples also inferred method development as part of the experimental studies. Investigating the role of BMAA in its producers was another purpose of the thesis, which may be crucial for future management of BMAA-producing cyanobacteria. By screening natural populations of the major filamentous bloom forming cyanobacteria of the Baltic Sea, we discovered the presence of BMAA throughout the entire summer season of two consecutive years, using a highly specific analytical method (liquid chromatography-tandem mass spectrometry; LC-MS/MS). BMAA was found to bioaccumulate in zooplankton and fish, as well as in mussels and oysters from the Swedish west coast. To improve the understanding of BMAA analyses in natural samples, the formation of carbamate adducts in the presence of bicarbonate was examined. Using two derivatization techniques in combination with LC-MS/MS, we could show that BMAA detection was not hindered by carbamate formation. Exogenously added BMAA inhibited nitrogen fixation in the model cyanobacterium Nostoc sp. PCC 7120, which was also hampered in growth and displayed signs of nitrogen starvation. Finally, BMAA was detected in cerebrospinal fluid in three of 25 Swedish test individuals, and represents the first confirmation of BMAA in the human central nervous system using LC-MS/MS as the primary analytical method. However, the association of BMAA to neurodegenerative diseases could not be verified as BMAA was present in both control individuals (two) and in one ALS-patient. Nevertheless, the finding of a known neurotoxic compound in the human central nervous system is alarming and potential consequences should be investigated. The discovery of the neurotoxic compound BMAA in Baltic Sea organisms, and in the central nervous system of humans potentially consuming fish from this ecosystem is concerning and warrants continued investigations of BMAA occurrence and human exposure. Further knowledge on the function and regulation of BMAA may help in developing strategies aiming to minimise human exposure. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2018-06-22T20:27:16Z No. of bitstreams: 2 Dissertação - Larissa Córdova Turones - 2018.pdf: 2576144 bytes, checksum: fb628d04d3221c6ac7ccaf79cc3d60ea (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Rejected by Jaqueline Silva (jtas29@gmail.com), reason: on 2018-06-22T20:38:02Z (GMT) / Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2018-06-22T20:39:29Z No. of bitstreams: 2 Dissertação - Larissa Córdova Turones - 2018.pdf: 2494325 bytes, checksum: 52754820438d7cd88e3dc298a96a898a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-06-27T11:25:24Z (GMT) No. of bitstreams: 2 Dissertação - Larissa Córdova Turones - 2018.pdf: 2494325 bytes, checksum: 52754820438d7cd88e3dc298a96a898a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-06-27T11:25:24Z (GMT). No. of bitstreams: 2 Dissertação - Larissa Córdova Turones - 2018.pdf: 2494325 bytes, checksum: 52754820438d7cd88e3dc298a96a898a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-05-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Bj-PRO-7a, a heptapeptide isolated and identified from Bothrops jararaca (Bj) venom, evoke potent therapeutic effects, as antihypertensive effect, natriuretic and diuretic effects, promotion of angiogenisis and vasodilatation. The effects of heptapeptide are independent of Angiotensin Converting Inhibitor (ACE), possibly dependent of Muscarinic Receptors subtype 1 (M1R) activation and oxido nitric pathways. Also, the Bj-PRO-7a actions upon central nervous system still need to be evaluated. Thus, the aims of this study were: i) to assess the effects of acute administration of Bj-PRO-7a upon behavior; ii) to reveal mechanisms involved in the effects of Bj- PRO-7a upon locomotion/exploration, anxiety and depression-like behaviors. For this purpose, adult male Wistar (WT) and Spontaneous Hypertensive Rats (SHRs) (300-380 g) received i.p. injections of Vehicle (NaCl 0.9%), Diazepam (2 mg/kg), Imipramine (15 mg/kg), Bj-PRO-7a (71, 213 or 426 nmol/kg), Pirenzepine (852 nmol/kg), α-metil-DL-tirosina (200 mg/kg) or Chlorpromazine (2 mg/kg) and were placed in the Elevated Plus Maze (EPM), Open Field (OF) and Forced Swimming (FS) tests. The heptapeptide promoted anxiolytic and antidepressantlike effects and increased the locomotion/exploration. These effects of Bj-PRO-7a seem to be strongly dependent on activation of M1R, catecholaminergic paths and dopaminergic receptors. / O Bj-PRO-7a, um heptapeptídeo isolado e identificado no veneno da Bothrops jararaca (Bj), evoca potentes efeitos terapêuticos, tais como o efeito anti-hipertensivo, efeitos natriurético e diurético, promoção da angiogênese e vasodilatação. Os efeitos do hepapeptídeo são independentes da inibição sobre a Enzima Conversora de Angiotensina (ECA), possivelmente dependentes da ativação dos Receptores Muscarínicos de Acetilcolina subtipo 1 (M1Rs) e vias do óxido nítrico. Ademais, as ações do Bj-PRO-7a sobre o sistema nervoso central ainda precisam ser avaliadas. Assim, os objetivos deste estudo foram: i) acessar os efeitos da administração aguda do Bj-PRO-7a sobre o comportamento; ii) revelar os mecanismos envolvidos nos efeitos do Bj-PRO-7a sobre a locomoção/exploração, comportamento tipo-ansiedade e depressão. Para esse propósito, ratos machos adultos Wistar (WT) e Espontaneamente Hipertensos (SHR) (300-380 g) receberam injeções i.p. de Veículo (NaCl 0,9%), Diazepam (2 mg/kg), Imipramina (15 mg/kg), Bj-PRO-7a (71, 213 ou 426 nmol/kg), Pirenzepina (852 nmol/kg), α-metil-DL-tirosina (200 mg/kg) ou Clorpromazina (2 mg/kg) e foram colocados nos testes de Labirinto em Cruz Elevado (LCE); Campo Aberto (CA) e Nado Forçado (NF). O heptapeptídeo promoveu efeito tipo-ansiolítico e antidepressivo e aumentou a locomoção/exploração. Esses efeitos parecem ser fortemente dependentes da ativação dos M1Rs, vias catecolaminérgicas e receptores dopaminérgicos.

Intrastriatal injections of L-Pyroglutamate (L-PGA) in mice produced behavioral and neuropathological effects that resemble in part the kainate-injected rat striatal model of Huntington's Disease (HD). The behavioral responses induced after unilateral injections of L-PGA included circling, postural asymmetry of head and trunk and possible dyskinesias. The neuropil in the injected striatum contained dilated profiles, degenerating neurons and oligodendroglia, and numerous phagocytic microglial-like cells. A dose response relation existed. The size of the lesion (expressed as a percent volume of the striatum destroyed) ranged from 1±0.18% at 0.02 μmoles to 20.2±3.97% at 200 μmoles L-PGA (pH=7.3). L-PGA is a weak neurotoxin when compared to kainic acid. Several factors raise interest in the possible role of L-PGA in HD, including the recently reported elevated plasma levels of L-PGA in some HD patients [51,52], and these are considered in the discussion.

Neurons depend upon neurotransmitter release through regulated exocytosis to accomplish the immense processing performed within the central nervous system. The SNARE hypothesis points to a family of proteins that are thought to enable the membrane fusion that leads to exocytosis. The secondary structure of SNAP-25 is unique among SNARE proteins in that it has two alpha helical SNARE motifs and a cysteine rich (C85, C88, C90, C92) membrane interacting region but notransmembrane domain. The cysteines may be modified by palmitoylation or oxidation but the role of these modifications in vivo is not well understood. Our goal is to elucidate possible regulatory roles of SNAP-25 that relate to its unique structure and these reversible modifications. However, the study of SNAP-25 in reconstituted systems is hampered by a lack of readily available palmitoylated SNAP-25. A method for in vitro palmitoylation of SNAP-25 by HIP14, a neuronal acyltransferase, is described along with the application of a biotinylation streptavidin assay to verify palmitoylation. Palmitoylation increases the extent to which SNAP-25 interacts with lipids as observed with an environment sensitive trpytophan fluorescence assay. Palmitoylation also alters the phase transition of DPPC lipids differently than unpalmitoylated SNAP-25.This effect on the membrane may influence fusion events. Oxidation of the cysteine residues may be responsible for the sensitivity of SNAP-25 to reactive oxygen species. Our data suggests that, when oxidized, SNAP-25 does not interact with membranes to the same extent as palmitoylated SNAP-25. This may provide a mechanism for reducing exocytosis during oxidative stress. Also, oxidized SNAP-25 is not susceptible to Botulinum Neurotoxin E. The effects of oxidation and palmitoylation on the protein interactions of SNAP-25 may shed light on its role in the SNARE complex and membrane fusion.

On a group level, satisfaction with botulinum neurotoxin (BoNT) treatment in neurological indications is high. However, it is well known that a relevant amount of patients may not respond as expected. The aim of this study is to evaluate the BoNT treatment outcome on an individual level using a statistical single-case analysis as an adjunct to traditional group statistics. The course of the daily perceived severity of symptoms across a BoNT cycle was analyzed in 20 cervical dystonia (CD) and 15 hemifacial spasm (HFS) patients. A parametric single-case autoregressive integrated moving average (ARIMA) time series analysis was used to detect individual responsiveness to BoNT treatment. Overall, both CD and HFS patients significantly responded to BoNT treatment with a gradual worsening of symptom intensities towards BoNT reinjection. However, only 8/20 CD patients (40%) and 5/15 HFS patients (33.3%) displayed the expected U-shaped curve of BoNT efficacy across a single treatment cycle. CD (but not HFS) patients who followed the expected outcome course had longer BoNT injection intervals, showed a better match to objective symptom assessments, and were characterized by a stronger certainty to control their somatic symptoms (i.e., internal medical locus of control). In addition to standard evaluation procedures, patients should be identified who do not follow the mean course-of-treatment effect. Thus, the ARIMA single-case time series analysis seems to be an appropriate addition to clinical treatment studies in order to detect individual courses of subjective symptom intensities.

Les neurotoxines botuliques (BoNTs), produites par C. botulinum, sont responsables du botulisme humain et animal. Dans sa forme naturelle, le botulisme résulte le plus souvent d’une absorption des toxines botuliques à partir du tube digestif après ingestion d’aliments contaminés par la toxine et C. botulinum. L’intoxination peut être divisée en 4 grandes étapes : production de toxine par la bactérie, ingestion d’aliments contenant la toxine préformée, passage de la neurotoxine à travers la barrière intestinale et action protéolytique aux terminaisons nerveuses. La régulation de la production des toxines et le passage des neurotoxines botuliques à travers la barrière intestinale sont mal compris. BoNT s’associe à des protéines non toxiques (NAPs) pour former des complexes de différentes tailles. Les gènes codant les BoNTs et NAPs sont regroupés sur le locus botulique et leur expression est contrôlée positivement par le facteur sigma alternatif BoTR/A. La toxinogénèse chez C. botulinum est contrôlée par un réseau complexe de régulateurs incluant au moins 3 systèmes à deux composants (TCS), identifiés pas la méthode d’ARN antisens, qui régulent positivement la production de complexe botulique indépendamment de BoTR/A. D’autre part, l’entrée de BoNT/B dans la barrière intestinale a été suivie à l’aide du fragment HcB marqué en fluorescence dans une anse intestinale ligaturée de souris. Des analyses en microscopie à fluorescence, immunohistochimie et microscopie électronique ont permis de mettre en évidence que HcB transcytose à travers les entérocytes par une voie d’endocytose dépendante de la dynamine. HcB cible les terminaisons nerveuses acétylcholinergiques de la lamina propia des villosités et gagne les neurones acétylcholinergiques et sérotoninergiques de la sous-muqueuse et de la musculeuse en seulement 10 minutes. Une étude in vitro réalisée sur cellules intestinales (m-ICcl2) montre que l’entrée de HcB est dépendante de récepteurs gangliosidiques GD1b/GT1b présents à la surface des cellules mais pas de la synaptotagmine II qui est requise pour l’entrée de BoNT/B dans les cellules neuronales. / Botulinum neurotoxins (BoNTs), produced by C. botulinum, are responsible for animal and human botulism. In its natural form, botulism is mostly acquired after absorption of BoNTs in the digestive tract after ingestion of food contaminated with C. botulinum and its toxins. The intoxination can be divided in 4 major steps: toxin production, ingestion of food contaminated with BoNTs, passage of BoNTs through the intestinal barrier, and proteolytic activity on nerve endings. Regulation of toxin production and passage of BoNTs through the intestinal barrier are poorly understood. BoNT associates with non toxic protein (NAPs) to form complexes of various sizes. The BoNTs and NAPs genes are clustered in the botulinum locus and are positively regulated by an alternative sigma factor BotR/A. Toxinogenesis in C. botulinum is regulated by a complex regulatory network containing at least 3 two components systems (TCS), identified by antisens RNA strategy, which regulate the production of botulinum complex independently of BotR/A. On the other hand, BoNT/B entry was monitored with fluorescent HcB fragment in ligatureted mouse intestinal loop. Fluorescent imaging analysis, immunohistochemistry and electron microscopy, have evidence that HcB is transcytosed through enterocytes cells by an endocytosis dynamin dependant. HcB targets acetylcholinergic nerves localized in lamina propria of villi then reaches serotoninergic and acetylcholinergic nerve endings in the submucosa and musculosa within 10 minutes. In vitro experiments performed on intestinal cell line (m-ICcl2) shows that the endocytosis of HcB is dependent on the GD1b/GT1b gangliosidic receptors on the cell surface but not on the synaptotagmine II protein which is recquiered HcB entry in neuronal cells

Les toxines botuliques (BoNTs) sont les substances les plus toxiques connues. Elles sont responsables du botulisme, une maladie rare mais le plus souvent mortelle sans prise en charge médicale. Cependant, les applications médicales des BoNTs sont de plus en plus nombreuses du fait de leurs propriétés paralysantes. Leur toxicité par voie inhalée en fait un des 6 principaux agents du risque intentionnel. Les BoNTs, produites par Clostridium botulinum, se répartissent en 7 types sérologiques qui se déclinent en sous-types. Cette biodiversité rend difficile leur identification par les méthodes classiques utilisées pour les toxines protéiques (approches immunologiques). Jusqu’à présent, seule l’analyse génétique permettait de distinguer les différents sous-types entre eux. Dans ce travail a été développée une méthode d’analyse en LC-QqQ-MS/MS en mode MRM pour identifier les différents sous-types de la BoNT/A dans des matrices complexes à partir de peptides communs et spécifiques à ces sous-types. Un traitement d’échantillon par immunocapture sur billes magnétiques couplées à des anticorps anti-peptides a été développé pour isoler la toxine de l’échantillon avant analyse. Des surnageants de culture des sous-types A1 à A3, A5, A7 à A8 ont été utilisés pour valider la méthode. La limite de détection de la méthode est compatible avec les taux de toxine retrouvés habituellement dans les échantillons naturellement contaminés. Cette méthode de spectrométrie de masse a ensuite été utilisée pour quantifier les différents sous-types de la BoNT/A dans une matrice complexe (surnageants de culture de C. botulinum). Une technique de quantification, utilisant un isotope stable de la chaine légère de type A1, ([13C6]K et [13C6]R), a été retenu comme étalon interne. Les différents sous-types de BoNT/A ont été quantifiés dans les surnageants et la quantité de BoNT correspondante à une dose létale minimale de 100% a été déterminée pour chaque sous-type. / Botulinum neurotoxins (BoNTs) are the most poisonous substances known. They are responsible for human botulism, a rare but potentially fatal disease if not quickly treated. However, BoNTs were approved for the treatment of numerous medical applications due to their temporary paralysis effects. BoNTs are among the six agents with the highest risk of potential use as bio-weapons because of their high toxicity in aerosol form. BoNTs, produced by Clostridium botulinum, are divised into seven toxinotypes and each toxinotype contains several subtypes. This biodiversity makes more difficult their identification with classical methods by immunological ways. Until now, only molecular genetical methods could differenciate subtypes among them. The aim of this work was to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) in MRM mode to efficiently discrimate the distinct subtypes from specific and common peptides. Immunocapture sample preparation with antipeptides antibodies was used and allowed the isolation of the toxin from the sample. Subtyping was performed with crude supernatants (BoNT/A1 to /A3, /A5, /A7 and /A8) in order to validate the method. Limit of detection (LOD) of the proposed method is in the range of minimal toxin concentration found in naturally contamined samples. In a second part of this work, this mass spectrometry method was used to quantify the neurotoxin in complex matrices (supernatants of Clostridium botulinum cultures). Isotope labeled light chain (13C6]K et [13C6]R) from botulinum A1 neurotoxin was produced and used as internal standart. Subtypes were quantified in supernatants and the quantity of neurotoxin for one minimal lethal dose 100% was determined for each subtype

Obwohl die Struktur und das Ausmaß dendritischer Verzweigungen eine wichtige Rolle bei der Informationsübertragung neuronaler Zellen spielen, ist bislang wenig über die Bausteine und Molekularmechanismen des Dendritenwachstums bekannt. Unter der Verwendung primär dissoziierter hippocampaler Zellkulturen embryonaler Mäuse untersuchte ich frühe Stadien des Zellfortsatzwachstums. Dabei konnte ich SNAP-25 (synaptosomal associated protein of 25 kDA), ein Schlüsselprotein der regulierten Exozytose, nicht nur in Axonen und terminalen Axonendigungen, sondern auch anhand von Doppelimmunmarkierungen mit den dendritischen Markern Transferrin-Rezeptor und MAP-2 in Dendriten lokalisieren. Die spezifische Inaktivierung von SNAP-25 durch Botulinumneurotoxin A (BoNT/A) führte zur Hemmung des Axonwachstums und des Vesikelrecyclings in terminalen Axonendigungen. Darüberhinaus wurde auch das Wachstum dendritischer Fortsätze von Körner- und Pyramidenzellen durch BoNT/A signifikant gehemmt. Daraus läßt sich schließen, daß SNAP-25, im Gegensatz zu Synaptobrevin, an konstitutiven Prozessen in den Axonen und Dendriten hippocampaler Neurone beteiligt ist. / Structure and dimension of the dendritic arbor are important determinants of information processing by the nerve cell, but mechanisms and molecules involved in dendritic growth are essentially unknown. I investigated early mechanisms of dendritic growth using mouse fetal hippocampal neurons in primary culture, which form processes during the first week in vitro. I detected a key component of regulated exocytosis, SNAP-25 (synaptosomal associated protein of 25 kDa)., in axons and axonal terminals as well as in dendrites identified by the occurrence of the dendritic markers transferrin receptor and MAP2. Selective inactivation of SNAP-25 by botulinum neurotoxin A (BoNTA) resulted in inhibition of axonal growth and of vesicle recycling in axonal terminals. In addition, dendritic growth of hippocampal pyramidal and granule neurons was significantly inhibited by BoNTA. These observations indicate that SNAP-25, but not synaptobrevin, is involved in constitutive axonal growth and dendrite formation by hippocampal neurons.

Snake venoms are a complex mixture of polypeptide and other molecules that adversely affect multiple homeostatic systems within their prey in a highly specific and targeted manner. Amongst the most potently toxic venoms in the world are those of the Australian venomous snakes, which belong almost exclusively to the elapid family. Their venoms posses a number of unique properties by which they target the mammalian cardiovascular and neuromuscular systems and are the focus for the identification of novel pharmacologically interesting compounds which may be of diagnostic or therapeutic benefit. Although much is known about the biochemical properties of Australia snake venoms as a whole, little research attention has focused upon individual components at the molecular level. This thesis describes the cloning, characterisation and comparative analysis of a number of unique toxins from the venom gland of the coastal taipan (Oxyuranus scutellatus) and a total of seven other related Australian snakes. These include the factor X- and factor V-like components of a prothrombin activator that causes a highly coagulable state in mammals. Comparative analysis of the sequences identified in this study, along with recombinant expression of an active form of the factor X-like component, provides important information on the structural, functional and evolutionary relationships of these molecules. Numerous other toxins were similarly identified and characterised including a pseudechetoxin-like protein, multiple phospholipase A2 enzymes and neurotoxin isoforms as well as vasoactive venom natriuretic peptides. Identified transcripts included not only toxin sequences but also other cellular peptides implicated in toxin processing, including a calglandulin-like protein. This thesis is the first description of the majority of these molecules at either the cDNA or protein level, and provides a means to study the activity of individual components from snake venoms and probe their function within the systems they specifically target. This study represents the most detailed and comprehensive description to date of the cloning and characterisation of different genes associated with envenomation from Australian snakes.