Dengue virus (DENV) is an arthropod-borne virus and has become a worldwide problem with steadily rising annual infection rates. Patients present with a range of symptoms from mild fever to, in some cases, life-threatening hemorrhagic fever and shock syndrome. The most severe cases require emergency hospital care and currently, there is no effective drug treatment or vaccine for dengue. As severe symptoms appear post-peak viremia, immuno-pathology is thought to be the cause and a potential trigger of this is differential activation of the immune response upon recognition of DENV. This could be due to a combination of factors including varying receptors, signalling pathways and immune regulation mechanisms. In order to understand DENV infection better, it is imperative to study the mechanisms of activation and control of immune responses triggered by the virus. Very early events in viral infection (after 10 min stimulation) were studied aiming to identify proteins involved in differential activation of immune responses. Phosphorylated proteins were isolated from cells post-stimulation and analysed by mass spectrometry. More than 200 proteins were differentially regulated by phosphorylation in response to DENV stimulation as compared to Mock, Influenza A virus and LPS stimulation. The effect of two specific proteins, namely Calpain-2 and Importin-5, identified to be differentially phosphorylated was investigated further. Calpain-2 was seen to be vital in the efficient production of progeny virions and the transcription of Mx1, an anti-viral interferon stimulated gene. Importin-5 is known to transport DENV NS5 into the nucleus during infection and was seen to co-precipitate with many host proteins. In summary, it is imperative that novel treatments and vaccines are developed for dengue as it is one of the world’s most prevalent arthropod-borne viruses. It was discovered here that many proteins undergo phosphorylation/de-phosphorylation in response to DENV stimulation to a differing degree than other stimuli. Calpain-2 plays a vital role DENV infection, potentially influencing the potency of immune response. Importin-5 associates with various host proteins during DENV infection, potentially altering their function or the function of Importin-5 itself. Research into targeted inhibition of Calpain-2 function or Importin-5 interaction with DENV NS5 could lead to a successful anti-viral treatment for DENV infection.

Infections by human schistosomes, in particular Schistosoma mansoni, account for significant morbidity and mortality every year in tropical and sub-tropical areas. The eggs of the parasite induce pathological changes in the infected host; in chronic and heavy infections, these changes may lead to death. A well-designed anti-schistosomal vaccine, alone or in concert with existing control measures such as chemotherapy, may prove to be a safe, inexpensive and effective means of reducing the occurrence of severe disease and death in S. mansoni infection. Previous studies have demonstrated the importance of the syncytial layer containing the apical plasma membrane (APM) of S. mansoni in both the survival of the parasite in the mammalian host and as a potential source of immunogens which may be utilized as vaccine candidates. In this paper we present evidence for the protective capacity of several schistosomal antigen preparations, including a calcium binding protein of the APM, S. mansoni calpain (GenBnnk accession no. M74233). We have constructed and characterized expression of a recombinant baculovirus expressing the large subunit of S. mansoni calpain, Sm-p80. This recombinant Sm-p80 is recognized by IgA, IgM, IgG1, and IgG3 isotype antibodies found in S. mansoni-infected human sera and partially-purified recombinant Sm-p80 provided a 29-39% reduction in worm burden in immunized mice challenged with S. mansoni. Our data indicate that Sm-p80 may be a useful vaccine antigen for the reduction of the morbidity associated with S. mansoni infections of mammalian hosts.

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Amyotrophic lateral sclerosis (ALS) is a devastating disease. Currently, there is no cure for this disease, and effective treatment strategies are greatly needed. Calpain activation plays a major role in the motor neuron degeneration that causes ALS. Therefore, therapeutic strategies can inhibit calpain activity in the central nervous system (CNS) have great clinical potential. The calpain inhibitors AK295 and MDL-28170 have been demonstrated to be neuroprotective in animal models of neurological injury, and should have great potential to treat ALS; however delivery problems have hindered their clinical success. Therefore, development of a new strategy that can locally deliver the calpain inhibitors to the central nervous system could significantly improve the treatment of ALS. The objectives of my thesis research were (1) to develop high molecular weight polyketals that provide sustained release properties for hydrophobic molecules, (2) to formulate calpain inhibitor-encapsulated polyketal microparticles which have a release half life of one month in vitro, (3) and to evaluate the performance of polyketal microparticles for delivering calpain inhibitors to the spinal cord in vivo. In completing these specific aims, we have developed biodegradable polymeric microparticles for the delivery of calpain inhibitors, AK295 and MDL-28170 to treat ALS. The results of calpain assays showed that both AK-PKMs and MDL-PKMs maintained most of their inhibitory activities even after the robust emulsion process. The in vitro release profile of MDL-28170 in MDL-PKMs showed that 50 % of the drug was released in the first 30 days. Experiments using dye-encapsulated microparticles showed that polyketal microparticles (1-2 ìm) are not easily cleared in the neutral physiological environment and can have potential to continuously release drug from the injection sites in the spinal cord. The efficacy of calpain inhibitor-encapsulated PKMs were studied by evaluation the behavior and survival of SOD1G93A rats, a genetic rat model for ALS. We observed the trend toward improvements in grip strength and rotarod performance in the first two months from the AK-PKMs treated group, however, further improvements are needed to enhance their in vivo efficacy.

In the hours and days following acute CNS injury, a secondary wave of events is initiated that exacerbate spinal tissue damage and neuronal cell death. A potential mechanism driving these secondary events is opening of the mitochondrial permeability transition pore (mPTP) and subsequent release of several cell death proteins. Previous studies have shown that inhibition of cyclophilin D(CypD), the key regulating component in mPTP opening, was protective against insults that induce necrotic cell death. We therefore hypothesized that CypD-null mice would show improved functional and pathological outcomes following spinal cord injury (SCI) and traumatic brain injury (TBI). Moderate and severe spinal contusion was produced in wild-type (WT) and CypD-null mice at the T-10 level using the Infinite Horizon impactor. Changes in locomotor function were evaluated using the Basso Mouse Scale (BMS) at 3 days post-injury followed by weekly testing for 4 weeks. Histological assessment of tissue sparing and lesion volume was performed 4 weeks post SCI. Calpain activity, measured by calpain-mediated spectrin degradation, was assessed in moderate injury only by western blot 24 hours post SCI. Results showed that following moderate SCI, CypD-null mice had no significant improvement in locomotor recovery or tissue sparing compared to wild-type mice. Following severe SCI, CypD-null mice showed significantly lower locomotor recovery and decreased tissue sparing compared to WT mice. Calpain-mediated spectrin degradation was not significantly reduced in CypD-null mice compared to WT mice 24h post moderate SCI. The lack of protective effects in CypD-null mice suggests that more dominant mechanisms are involved in the pathology of SCI. In addition, CypD may have a pro survival role that is dependent on the severity of the spinal cord injury.

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Rôle du Système de calpaïne/calpastatine dans l'hypertension pulmonaire induite par l'hypoxie chez la souris. Les souris calpaïnes knockout ont montré des effets protecteurs dans l'hypertension pulmonaire (HP) induite par l'hypoxie. Cependant, le modèle animal avec une surexpression de calpastatine (cast) n'a jamais été étudié. Notre objectif est d'utiliser des souris transgéniques CMV-cast qui surexpriment constitutivement la calpastatine intracellulaire sous le contrôle du promoteur CMV dans tous les types cellulaires pour étudier les effects de la calpaïne intracellulaire. Nous utilisons aussi des souris transgéniques CRP-cast qui surexpriment la calpastatine extracellulaire sous le contrôle du promoteur CRP (protéine C-réactive) pour étudier les effets de la calpaïne extracellulaire. Finalement, nous examinons les effets d'un traitement avec le PD150606, inhibiteur de calpaïne, chez des souris C57BL/6j (WT) hypoxiques et SM22-5HTT+ qui dévélopent l'HP spontanément. Nous avons constaté que les protéines calpaïne et calpastatine sont augmentées immédiatement dans un état hypoxique. Les calpaïnes ont ensuite culminé le jour 8 et sont restées élevées jusqu'au jour 18 chez les souris WT hypoxiques, alors que la calpastatine a augmenté du jour 1 au jour 3, retournant au niveau basal jusqu'au jour 18. Les activités des calpaïnes intra- et extra-cellulaires ont augmenté progressivement pour atteindre un sommet au jour 8, restant aux niveaux élevés jusqu'au jour 18 chez les souris WT hypoxiques. En utilisant l'immunofluorescence, nous avons constaté que l'augmentation des calpaïnes sont principalement colocalisés avec les CML vasculaires pulmonaires (α-SMA+). Cependant, chez la souris CMV-cast, la surexpression de la calpastatine a atténué le développement d'HP. Chez les souris CMV-cast hypoxiques les niveaux de calpastatine sont restés plus élevés que ceux des souris WT à tous les moments de l'hypoxie. Les niveaux de calpastatine plus élevés chez les souris CMV-cast ont empêché de manière significative une augmentation des niveaux de protéines calpaïnes et des activités intra- et extra-cellulaires de la calpaïne au cours de l'hypoxie. Les résultats d'immunofluorescence également ont confirmé que moins de calpaïnes colocalisent avec les CML vasculaires pulmonaires (α-SMA+) chez la souris CMV-cast. Après 18 jours hypoxie, CRP-cast mice ont attenué le développement d'HP. En outre, cette surexpression a montré des effets similaires par rapport à celle intracellulaire. Cependant, le PD150606 a eu que des effets supplémentaires chez les souris WT hypoxiques par rapport aux souris CMV-cast et CRP-cast hypoxiques. Chez les souris SM22-5HTT+, les niveaux de calpastatine, de calpaïnes ainsi que des activités intra- et extra-cellulaires de la calpaïne ont été significativement augmentés dans les poumons. Le PD150606 n'a pas modifié les niveaux de calpastatine, mais il a diminué de manière significative des calpaïnes ainsi que des activités intra- et extra-cellulaire de calpaïne. Les niveaux de calpastatine et des calpaïnes ont également paru augmentées dans les vessaux pulmonaires remodelés chez les patients atteints de maladie pulmonaire chronique par rapport à ceux nonremodelés. En résumé, nos résultats indiquent un nouveau rôle des calpaïnes extracellulaires dans la prolifération des CML-AP dans l'HP. Les stratégies d'inhibition des calpaïnes extracellulaires sembleraient être une stratégie thérapeutique dans le traitement de la progression de l'HP. / Targeting the Calpain/Calpastatin System to Protect against Hypoxia-induced Pulmonary Hypertension in Mice. Calpain knockout mice exhibited protective effects against hypoxia-induced PH. Our aim was to study the role of the calpain/calpastatin system on PH development in mice. To this end, we used mice ubiquitously overexpressing intracellular calpastatin (cast) under the control of a CMV promoter (CMV-cast) to explore the effects of intracellular calpains. We also used mice overexpressing extracellular calpastatin under the control of a CRP (C-reactive protein) promoter (CRP-cast) to explore the effect of extracellular calpains. Finally, we examined the effects of treatment with PD150606, an inhibitor of calpain, in WT mice exposed to hypoxia and in SM22-5HTT+ mice with spontaneous PH. During time-course of hypoxia, we found that calpain and calpastatin protein levels increased immediately after hypoxic exposure. Calpain protein levels then peaked on day 8 and remained elevated until day 18 in hypoxic WT mice; however, calpastatin protein levels increased from day 1 to day 3, and returned to basal level until day 18. Both intra- and extra-cellular calpain activities were upregulated gradually and peaked on days 8, and still markedly remained in high levels until day 18 in hypoxic WT mice. By using immunofluorescence, we found that increased calpains were predominantly colocalized with α-SMA positive pulmonary vascular SMCs. In CMV-cast mice, intracellular calpastatin overexpression successfully attenuated PH development. In CMV-cast mice, calpastatin protein levels remained higher than those in WT mice at all time points of hypoxia. The higher calpastatin protein levels in CMV-cast mice did significantly prevent an increase in calpain protein levels and calpain intra- and extra-cellular activities during hypoxia. After 18 days hypoxia, CRP-cast mice exhibited less PH severity. Moreover, extracellular calpastatin overexpression showed similar effects as intracellular calpastatin overexpression. Treatment with PD150606 induced an additional protective effect in hypoxic WT mice but not CMV-cast and CRP-cast mice. In SM22-5HTT+ mice, lung calpastatin and calpain proteins as well as calpain intra- and extra-cellular activities were significantly increased. PD150606 did not alter lung tissue calpastatin. However, it significantly decreased calpain protein levels as well as calpain intra- and extra-cellular activities. In summary, our present results demonstrate that calpain inhibition prevents PH development. Either increasing extracellular calpastatin or increasing both extra- and intra-cellular calpastatin is efficient to attenuate PH. Treatment with PD150606 which inhibits both extra- and intra-cellular calpain activities may be useful in teh setting of PH.

Os agonistas beta-adrenérgicos (βAA) são conhecidos por aumentar a hipertrofia muscular e lipólise, neste caso uma maneira de se reduzir o efeito da lipólise seria a imunocastração. Dessa forma, o objetivo deste projeto foi avaliar o efeito dos βAA e da imunocastração sobre a qualidade da carne de bovinos Nelore. Foram utilizados noventa e seis bovinos Nelore, sendo que metade dos animais (n=48) receberam uma dose da vacina de imunocastração, e após 30 dias receberam a segunda dose. A outra metade dos animais (n=48) não recebeu nenhuma dose da vacina. Durante 70 dias os animais foram alimentados com uma dieta padrão composta de 24% volumoso e 76% de concentrado. Após 70 dias de confinamento os animais foram divididos em três grupos, dentro de bloco (peso inicial) e condição sexual e foram alimentados por 30 dias, com umas das seguintes dietas: CON - dieta padrão utilizada na fase anterior, sem a adição de βAA; ZIL - dieta padrão acrescida de 80 mg/dia Cloridrato de Zilpaterol; RAC - dieta padrão acrescida de 300 mg/dia Cloridrato de Ractopamina. Ao final desse período os animais foram abatidos e colhidas amostras do músculo Longissimus dorsi para as avaliações de qualidade de carne, lipídeos totais, perfil de ácidos graxos, análise sensorial do consumidor, perfil morfométrico muscular, expressão dos genes calpaína e calpastatina, comprimento de sarcômero. Para a maioria das características avaliadas não foram observadas interações entre os tratamentos. Ao avaliar o efeito da condição sexual, os animais imunocastrados apresentaram maiores intensidades de cor L, a e b, lipídios totais, ácidos oleico, palmítico e total de monoinsaturados e maior frequência para as fibras oxidativas (FO) e glicolíticas (FG) em relação aos não-castrados. Contudo, os animais não-castrados tiveram uma tendência a apresentarem uma carne mais macia na análise sensorial e obtiveram maior frequência das fibras oxidativasglicolíticas (FOG) em relação aos imunocastrados. Quanto ao efeito dos βAA, o grupo ZIL apresentaram uma carne menos macia na força de cisalhamento, maiores concentrações de ácidos heptadecanoico, linoleico, araquidonico ácido C20:3 N6C8C11C14, ômega 6, maior frequência para as FO e menor para FG em comparação ao grupo RAC e CON. No entanto, os animais do grupo CON e ZIL apresentaram maior área para as FO em comparação ao grupo RAC, enquanto que para as FOG, os animais do grupo CON tiveram maior área do que os animais do grupo RAC e ZIL. Na análise sensorial, os grupos RAC e ZIL receberam menores notas para os atributos textura e qualidade global em relação ao CON. Não foi observado efeito da condição sexual e dos βAA sobre a expressão dos genes e comprimento de sarcômero. Conclui-se que a condição sexual e a suplementação com os βAA podem alterar a qualidade da carne, perfil de ácidos graxos e morfométrico muscular, sem, contudo, alterar a expressão dos genes e do comprimento de sarcômero. / The Beta adrenergic agonist (βAA) are knowed for increase muscle hypertrophy and lipolysis, in this case on way for decrease the lipolysis effect is use the immunocastration. The objective of this research was evaluated the effect of βAA and immunocastration on meat quality of Nellore . Ninety-six Nellore were fed in this trial; half of the animals (n = 48) received one dose of immunocastration vaccine on d 0, and received another dose at d 30. The other half of animals (n = 48) received no vaccine. Animals were fed with a standard diet consisting of 24% forage and 76% concentrate for 70 d. After 70 d of the standard diet, animals were divided into three groups, and were fed 30 d with one of the following diets: CON - standard diet used in the previous phase, without the addition of βAA; ZIL - standard diet plus 80 mg/d Zilpaterol hydrochloride; RAC - standard diet plus 300 mg/d Ractopamine hydrochloride. After this period, animals were harvested and the Longissimus dorsi sample were colleted to evaluate meat quality, total lipid content, fatty acid profile, consumer sensory analysis, muscle morfometric profile, genes expression of calpain and calpastatin and sarcomere length. For almost of characteristics evaluated, were not observed interactions between treatments. The effect of sexual condition, imunocastrated animals showed higher intensity of color L, a and b, total lipidics, oleic, palmitic and total monounsaturated acids and more frequency for oxidative fibers (FO) and glycolytic fibers (FG) in relation at noncastrated. However, non-castrated animals had a tendency to show a meat tender in sensory analysis and more frequency of oxidative-glicolytics fibres (FOG) in relation to imunocastrated. The βAA effect, ZIL group showed a meat less tender, higher concentrations of heptadecanoic, linoleic, araquidic acids, C20:3 N6C8C11C14, ômega 6, higher frequency for FO and less for FG than RAC and CON group. Animals of CON and ZIL group showed more FO area than RAC group, while for the FOG, animals from COM group showed more area than animals from RAC and ZIL group. In the sensory analysis, RAC and ZIL group received lower grades for tenderness and global quality in relation to COM group. Was no observed effect of sexual condition and βAA for genes expressions and sarcomere length. As conclusion, sexual condition and βAA affected the meat quality, fatty acid profile, muscle fibers, but not affect genes expression and sarcomere lenght.