A Austrália é um país cuja fauna é um repositório de potenciais novos biofármacos, pois se encontram no continente os animais mais mortais do planeta, dentre eles, as serpentes. A serpente Pseudechis australis (Mulga snake) é a maior serpente venenosa da Austrália e tem ampla distribuição geográfica. Os venenos de serpentes são complexas misturas com proteínas e peptídeos que apresentam uma variedade de atividades biológicas. Devido à riqueza de seus componentes, várias moléculas encontradas no veneno vêm sendo utilizadas com fins terapêuticos, como agentes anticoagulantes ou analgésicos. Apesar dessas informações, existem poucos dados disponíveis sobre os componentes específicos deste veneno. O presente trabalho tem como objetivo isolar e caracterizar as proteases desse veneno, ainda não descritas, um primeiro passo para compreender o papel destas enzimas no processo de envenenamento, assim como seus inibidores endógenos. Estes desempenham uma função protetora da glândula de veneno, inibindo a ação das enzimas in loco, prevenindo assim a degradação do tecido glandular por estas toxinas. O interesse nestes inibidores está relacionado ao seu potencial uso na terapia de diversas doenças como distúrbios da coagulação, hipertensão e câncer. / Australia is a natural repository of some of the deadliest venomous animals on the planet and, as such, a potential source for new toxin-derived drugs. Venomous snakes are among the many potential sources of new promising compounds. Snake venoms are complex mixtures of proteins and peptides that exhibit a variety of biological activities which all are directed towards subduing the prey and/or aggressor. These toxins act disturbing homeostasis, affecting neural transmission, hemostasis, tissue integrity as well as other body functions. Such a a wide array of specific activities has turned snake toxins into successful drugs used for therapeutic purposes, as anticoagulants or analgesic agents. Unlike snake venoms from other parts of the world, there are few records on the venom composition of Australian snakes, turning these into potential sources of new bioactive molecules for drug design. This study aims to isolate and characterize the yet undescribed proteases of the venom of P. australis as well as their endogenous inhibitors, as a first step in understanding the role of these enzymes in the envenoming process.

A Austrália é um país cuja fauna é um repositório de potenciais novos biofármacos, pois se encontram no continente os animais mais mortais do planeta, dentre eles, as serpentes. A serpente Pseudechis australis (Mulga snake) é a maior serpente venenosa da Austrália e tem ampla distribuição geográfica. Os venenos de serpentes são complexas misturas com proteínas e peptídeos que apresentam uma variedade de atividades biológicas. Devido à riqueza de seus componentes, várias moléculas encontradas no veneno vêm sendo utilizadas com fins terapêuticos, como agentes anticoagulantes ou analgésicos. Apesar dessas informações, existem poucos dados disponíveis sobre os componentes específicos deste veneno. O presente trabalho tem como objetivo isolar e caracterizar as proteases desse veneno, ainda não descritas, um primeiro passo para compreender o papel destas enzimas no processo de envenenamento, assim como seus inibidores endógenos. Estes desempenham uma função protetora da glândula de veneno, inibindo a ação das enzimas in loco, prevenindo assim a degradação do tecido glandular por estas toxinas. O interesse nestes inibidores está relacionado ao seu potencial uso na terapia de diversas doenças como distúrbios da coagulação, hipertensão e câncer. / Australia is a natural repository of some of the deadliest venomous animals on the planet and, as such, a potential source for new toxin-derived drugs. Venomous snakes are among the many potential sources of new promising compounds. Snake venoms are complex mixtures of proteins and peptides that exhibit a variety of biological activities which all are directed towards subduing the prey and/or aggressor. These toxins act disturbing homeostasis, affecting neural transmission, hemostasis, tissue integrity as well as other body functions. Such a a wide array of specific activities has turned snake toxins into successful drugs used for therapeutic purposes, as anticoagulants or analgesic agents. Unlike snake venoms from other parts of the world, there are few records on the venom composition of Australian snakes, turning these into potential sources of new bioactive molecules for drug design. This study aims to isolate and characterize the yet undescribed proteases of the venom of P. australis as well as their endogenous inhibitors, as a first step in understanding the role of these enzymes in the envenoming process.

Evolution can occur rapidly, along timescales that are traditionally regarded as 'ecological'. Despite growing acceptance among biologists of rapid evolution, a strong paradigm of contemporary evolution is still absent in many sub-disciplines. Here I apply a contemporary evolution viewpoint to conservation biology. Specifically, I examine the impact of cane toads (Bufo marinus) on Australian snakes. Toads were introduced into Australia in 1935, have spread rapidly and represent a novel, extremely toxic prey item to na�ve Australian predators (including snakes). Based on dietary preferences and geographic distributions I find that 49 species of Australian snake are potentially at risk from the invasion of the toad. Furthermore, examination of physiological resistance to toad toxin in 10 of these �at risk� species strongly suggests that most species of Australian snake are poorly equipped to deal with a likely dose of toad toxin. Even species that are highly resistant to toad toxin (such as the keelback, Tropidonophis mairii) face indirect fitness costs associated with consuming toads. Within a population of snakes however, the impact of toads is unlikely to be random. For example, the examination of several component allometries describing the interaction between snakes and toads revealed that, within a species, smaller snakes are more likely to ingest a fatal dose of toad toxin than are larger snakes. Further consideration of the interaction between snakes and toads suggests that toads will not only be exerting differential impact on snakes based upon morphology, but also exert non-random selection on prey preference and resistance to toad toxin in snake populations. To examine the possibility of a morphological response by snakes to toads, I examined changes in the body size and relative head size of four species of snake as a consequence of time since exposure to toads. Two of the species (green treesnakes and red-bellied blacksnakes) are predicted to face strong impacts from toads. These two species showed an increase in mean body size and a decrease in relative head size as a consequence of time since exposure to toads; both changes in an adaptive direction. In contrast, the other two species (keelbacks and swampsnakes) are predicted to face much lower impact from toads, and these two species showed little or no evidence of morphological change associated with time since exposure to toads. These results indicate an adaptive change in morphology at a rate that is proportional to the predicted level of impact for each species, strongly suggesting an evolved response. Red-bellied blacksnakes (a toad-vulnerable species) were further assessed for evolved responses in prey preference and toxin resistance. Comparisons between toad-exposed and toad-na�ve populations of blacksnakes revealed that snakes from toad-exposed populations exhibited slightly higher resistance to toad toxin and a much-reduced tendency to eat toads, when compared with toad-na�ve snakes. Na�ve snakes exhibited no tendency to learn avoidance of toxic prey, nor were they able to acquire resistance to toxin as a result of several sub-lethal doses, suggesting that the observed differences between populations is evolved rather than acquired. Together, these results strongly suggest that blacksnakes are exhibiting an evolved shift in prey preference and toxin resistance as a consequence of exposure to toads. Thus, it appears that snakes are exhibiting adaptation at multiple traits in response to exposure to toads. Given the high likelihood that these adaptive shifts have an evolved basis, it appears that the impact of toads will decrease with time in many snake populations. But what about toads? Because the outcome of the interaction between a toad and a snake is also mediated by the body size and relative toxicity of toads, it is important to understand how these traits vary in space and time. Exploratory analysis revealed that toads exhibit a decrease in body size and a decrease in relative toxicity as a consequence of time since colonisation, indicating that their impact on native predators decreases with time. Additionally, there appears to be meaningful spatial variation in toad relative toxicity, indicating that some populations of native predators are facing higher impact from toads than others. Overall, these results clearly indicate the importance of assessing the potential for rapid evolutionary response in impacted systems. Doing so may provide evidence that some species are in less trouble than originally thought. Additionally, and as more data accumulate, it may be possible to characterise certain categories of environmental impact by their potential for eliciting adaptive response from �impacted� species. This approach has strong implications for the way conservation priorities are set and the way in which conservation dependent populations are managed.

Evolution can occur rapidly, along timescales that are traditionally regarded as 'ecological'. Despite growing acceptance among biologists of rapid evolution, a strong paradigm of contemporary evolution is still absent in many sub-disciplines. Here I apply a contemporary evolution viewpoint to conservation biology. Specifically, I examine the impact of cane toads (Bufo marinus) on Australian snakes. Toads were introduced into Australia in 1935, have spread rapidly and represent a novel, extremely toxic prey item to na�ve Australian predators (including snakes). Based on dietary preferences and geographic distributions I find that 49 species of Australian snake are potentially at risk from the invasion of the toad. Furthermore, examination of physiological resistance to toad toxin in 10 of these �at risk� species strongly suggests that most species of Australian snake are poorly equipped to deal with a likely dose of toad toxin. Even species that are highly resistant to toad toxin (such as the keelback, Tropidonophis mairii) face indirect fitness costs associated with consuming toads. Within a population of snakes however, the impact of toads is unlikely to be random. For example, the examination of several component allometries describing the interaction between snakes and toads revealed that, within a species, smaller snakes are more likely to ingest a fatal dose of toad toxin than are larger snakes. Further consideration of the interaction between snakes and toads suggests that toads will not only be exerting differential impact on snakes based upon morphology, but also exert non-random selection on prey preference and resistance to toad toxin in snake populations. To examine the possibility of a morphological response by snakes to toads, I examined changes in the body size and relative head size of four species of snake as a consequence of time since exposure to toads. Two of the species (green treesnakes and red-bellied blacksnakes) are predicted to face strong impacts from toads. These two species showed an increase in mean body size and a decrease in relative head size as a consequence of time since exposure to toads; both changes in an adaptive direction. In contrast, the other two species (keelbacks and swampsnakes) are predicted to face much lower impact from toads, and these two species showed little or no evidence of morphological change associated with time since exposure to toads. These results indicate an adaptive change in morphology at a rate that is proportional to the predicted level of impact for each species, strongly suggesting an evolved response. Red-bellied blacksnakes (a toad-vulnerable species) were further assessed for evolved responses in prey preference and toxin resistance. Comparisons between toad-exposed and toad-na�ve populations of blacksnakes revealed that snakes from toad-exposed populations exhibited slightly higher resistance to toad toxin and a much-reduced tendency to eat toads, when compared with toad-na�ve snakes. Na�ve snakes exhibited no tendency to learn avoidance of toxic prey, nor were they able to acquire resistance to toxin as a result of several sub-lethal doses, suggesting that the observed differences between populations is evolved rather than acquired. Together, these results strongly suggest that blacksnakes are exhibiting an evolved shift in prey preference and toxin resistance as a consequence of exposure to toads. Thus, it appears that snakes are exhibiting adaptation at multiple traits in response to exposure to toads. Given the high likelihood that these adaptive shifts have an evolved basis, it appears that the impact of toads will decrease with time in many snake populations. But what about toads? Because the outcome of the interaction between a toad and a snake is also mediated by the body size and relative toxicity of toads, it is important to understand how these traits vary in space and time. Exploratory analysis revealed that toads exhibit a decrease in body size and a decrease in relative toxicity as a consequence of time since colonisation, indicating that their impact on native predators decreases with time. Additionally, there appears to be meaningful spatial variation in toad relative toxicity, indicating that some populations of native predators are facing higher impact from toads than others. Overall, these results clearly indicate the importance of assessing the potential for rapid evolutionary response in impacted systems. Doing so may provide evidence that some species are in less trouble than originally thought. Additionally, and as more data accumulate, it may be possible to characterise certain categories of environmental impact by their potential for eliciting adaptive response from �impacted� species. This approach has strong implications for the way conservation priorities are set and the way in which conservation dependent populations are managed.

Os venenos de serpentes peçonhentas representam uma mistura muito rica em compostos orgânicos e inorgânicos com potencial farmacológico, clínico e médico considerável. (As propriedades de seus componentes vêm sendo pesquisadas e características que antes não eram observáveis passam a ser descobertas influenciando à obtenção de novas drogas e na otimização de terapias já existentes. A maior parte dos compostos presentes nos venenos das serpentes é formada por proteínas que representam de 90 a 95% do peso seco do veneno, dentre elas destacam-se as peptidases (serino e metalopeptidases). As serinopeptidases caracterizam-se por apresentar no sítio ativo uma tríade catalítica idêntica a presente na tripsina, a qual é formada pelo agrupamento dos aminoácidos, His 57, Asp 102 e Ser 195. Constituem um grupo de enzimas que apresentam uma ampla variedade de efeitos biológicos, com massa molecular entre 25 kDa e 35 kDa, promovendo diversos efeitos fisiológicos como, alterações no sistema hemostático, agindo nos componentes da cascata de coagulação e com ação fibrinogenolítica. Ainda que já existam enzimas de veneno de serpentes com aplicação na regulação da cascata da coagulação, vários laboratórios têm buscado novas alternativas que apresentem atividade na hemostasia a fim de obtenção de produtos farmacológicos ainda não investigados. As serpentes australianas da família Elapidae, destacam-se para este fim, por apresentarem proteínas pró-coagulantes e até o momento não há descrição na literatura sobre a presença de serinopeptidases no veneno de Pseudechis australis. Neste trabalho conseguimos isolar e confirmar pela primeira vez a presença de uma serinopeptidase do veneno de Pseudechis australis através de técnicas cromatográficas e bioquímicas. Esta serinopeptidase possui atividade sobre a cadeia beta e alfa do fibrinogênio, não possui atividade gelatinolítica e possui semelhanças com o Fator X (FX) da cascata de coagulação. / Snake venoms represent a very rich mixture of organic and inorganic compounds with potential pharmacological, clinical and medical applications. This potential has not been fully exploited due to our incomplete knowledge of the properties of venoms and their components. Most of the compounds present in snake venoms are proteins that represent 90 to 95% of the dry weight, among which stand out peptidases (serine and metallopeptidases). The serinopeptidases are characterized by having a catalytic triad similar to the one present in trypsin, which is formed by, His 57, Asp 102 and Ser 195.These proteins constitute a group of enzymes that have a variety of activities, molecular mass between 25 kDa to 35 kDa, promoting many physiological effects as changes in the hemostatic system, acting on components of the clotting cascade and fibrinogenolytic action. Several snake venoms isolated peptidases are used for therapeutic purposes, among which serinopeptidases as Batroxobin® and Ancrod® used as anticoagulants in heart surgery. Pseudechis australis (King Brown or Mulga Snake) is a terrestrial species among the most poisonous of Australia and belonging to the family Elapidae which constitutes 57.7% of the snake species described in the Australian continent, Australian Elapidae are among the ten more toxic species in the world. Although there are already venom enzymes snakes with application in coagulopathies, several laboratories have sought alternatives for the treatment of hemostasic disorders in order to obtain new pharmacological products. Australian snakes from the Elapidae family, stand out for this purpose, because they have procoagulant proteins with properties similar to the macromolecular complex found in blood. It is worth noting that so far there is no description in the literature on the presence of serinopeptidases in Pseudechis australis venom and since serinopeptidases are closely linked to the hemostatic system, new serinopeptidases may have great potential in the development of drugs for the therapy of hemostatic disorders. In this work we managed to isolate and confirm the first time a serinopeptidase the Pseudechis australis venom through chromatographic and biochemical techniques. This serinopeptidase have activity on alpha and beta chain of fibrinogen, has no gelatinolytic activity and has similarities to factor X of the clotting cascade.