The complete cDNA sequence of a mitochondrial protein from Chinese hamster ovary cells has been determined. This protein, designated P1, was originally identified in cells resistant to the microtubule inhibitor podophyllotoxin (Gupta, 1981). The mutant cell line contained an alteration of the P1 protein that gave rise to a new, more acidic protein, designated M1 (Gupta et al., 1982). The P1 protein was determined to be microtubule-related based on the cross-resistance pattern of the mutants to other microtubule inhibitors, and corelease with tubulin under conditions which cause microtubule depolymerization (Gupta et al., 1982). Subcellular fractionation studies localized this protein to the matrix of the mitochondria (Gupta and Austin, 1987). Antibodies raised against P1 were used to isolate a cDNA clone from human cells (Jindal et al.,1989). The human cDNA clone was used as a probe to screen for clones of the P1 protein in bacteriophage (lambda)gt10/(lambda)gt11 cDNA libraries prepared from CHO cells. The P1 cDNA encodes a protein of 573 amino acids with a relative molecular mass of 60,983 daltons. The first 26 amino acids meet the requirements of a mitochondrial matrix targeting sequence. The mature protein is 547 residues in length with a relative molecular mass of 57,949 daltons. The deduced amino acid sequence shows 97% identity to the the human P1 protein. More interestingly, the amino acid sequence shows extensive homology (42 to 55% identical residues and an additional 15 to 20% conservative replacements) to the chaperonin class of molecular chaperones. This class of proteins includes the hsp60 protein of yeast, the groEL protein of Escherichia coli, the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit binding protein of plant chloroplasts, and the 62-65-kDa major antigenic protein of mycobacteria and Coxiella burnetii. The homology of P1 with the above proteins begins after the putative mitochondrial presequence and extends to the c-terminal end. Several regions throughout the protein sequence are highly conserved and are proposed to be functional domains of the protein. Also highly conserved is a Gly-Gly-Met repeating motif at the carboxy-terminus. The function of this sequence is undetermined, as yet. A dendrogram was constructed from the sequence homology data. It suggested that mitochondrial P1 evolved from purple bacteria which is the endosymbiont which gave rise to mitochondria. The chaperonin class of proteins have been shown to assist in the assembly of oligomeric protein structures. It is suggested that the P1 protein may play a similar role in mammalian cells. The high degree of homology between P1 and the 65-kDa mycobacterial antigen also suggests that P1 may be involved in certain autoimmune diseases. / Thesis / Master of Science (MS)

No description available.

O emprego do hamsters (Mesocricetus auratus) como modelo biológico experimental para a reprodução da condição de portador renal de leptospiras foi investigado em machos e fêmeas jovens com 80 a 120 g de peso vivo. Os animais foram experimentalmente infectados com estirpe patogênica do sorovar Pomona caracterizada por provocar a morte por leptospirose entre o quinto e o décimo dia pós-infecção. No segundo dia pós-infecção os animais foram tratados com estolato de eritromicina, nas concentrações de 10, 20, 40 e 80 mg/kg de peso vivo. Aos 30 dias, da infecção experimental os sobreviventes foram anestesiados com isofluorano e procedeu-se a colheita de sangue para a determinação dos indicadores da função hepática e renal (Proteínas totais, Albumina, Uréia, Creatinina, Fosfatase Alcalina, Alanina Aminotransferase, Aspartato Aminotransferase, Bilirrubinas Indireta, Bilirrubinas Direta e Bilirrubinas Totais), bem como o titulo de aglutininas pela prova de soro aglutinação microscópica. A seguir, com o aprofundamento da anestesia, os animais foram submetidos a eutanásia e necropsiados para a colheita de tecido renal e hepático destinados aos exames histopatológicos pelas colorações de Hematoxilina - Eosina e Warthin- Starry, bem como do isolamento de leptospiras por cultivo em meio de Fletcher. Houve controles do inóculo infeccioso, do tratamento com antibiótico e do sistema de manejo adotado. O número de DL 50 efetivamente empregadas no inóculo infeccioso foi de 7,11. No grupo controle do antibiótico foi constatado elevação do nível de fosfatase alcalina e degeneração vacuolares dos hepatócitos para as concentrações de 40 a 80 mg de antibiótico. Os portadores renais de leptospira foram obtidos entre os animais tratados com 40 ou 80 mg de estolato de eritromicina, independentemente do sexo; estes animais apresentaram elevação dos níveis séricos de creatinina e proteínas totais já as determinações de albumina, uréia, alanina aminotrasferase, aspartato aminotransferase, bilirrubinas direta, bilirrubinas indiretas e totais foram iguais as encontradas em animais não infectados por leptospiras e não tratados com antibióticos. As alterações histológicas encontradas nos animais portadores de leptospiras foram degeneração vacuolar em hepatócitos, sangue no espaço porta, congestão glomerular. Nos animais induzidos a condição de portadores renais de leptospiras os títulos de anticorpos aglutinantes, para o sorovar homólogo ao da infecção, expressos em logaritmo de base, 10 foram iguais ou superiores a 1,19. / The use of hamsters (Mesocricetus auratus) as experimental model for the reproduction of leptospires kidney carrier condition was investigated on youngs males and females with 80 to 120 g of living weight. The animals were experimentally infected with pathogenic strain of serovar Pomona able of causing the death by leptospirosis between the fifth and the tenth post-infection day. On the second day post-infection the animals were treated with erythromycin estolate at the concentrations of 10, 20, 40 and 80 mg/ kg of living weight. At the 30th day of experimental infection the survivors were anesthetized with isoflurane and blood sample were collected for the determination of kidney and liver functions (Total proteins, Albumin, Urea, Creatinine, Alkaline Phosphatase, Alanine Aminotransferase, Aspartate Aminotransferase, Indirect Bilirubins, Direct Bilirubins and Total Bilirubins), and the of agglutinine title by the microscopic agglutination test (MAT). The animals were killed whit the reinforcement of the anesthesis, and necropsied for the collection of kidney and liver sample for histopathologic tests by staining of Hematoxylin-Eosina and Warthin-Starry, such as the isolation of leptospiras by cultivation into Fletcher´s medium. There were controls of the infecctious inoculum, antibiotic treatment and of the management system adopted. The number of DL 50 effectively applied in the infectious inoculum was 7,11. The antibiotic controls presented elevation of the alkaline phosphatase level and vacuolar degeneration of hepatocytes at the concentrations of 40 to 80 mg of antibiotic. The leptospire\'s kidney carriers were obtained in the animals treated with 40 or 80 mg of Erythromycin Estolate, regardless of the sex; these animals showed increase in creatinine\'s and total protein serum levels but of albumin, urea, alanine aminotransferase, aspartate aminotransferase, direct bilirubins, indirect bilirubins and total bilirubins were the same as found in animals not infected by leptospires and not treated with the antibiotic. The histological changes found in the animals induced as leptospires carrier were vacuolar degeneration in hepatocytes, blood in the portal tract, and glomerular congestion. The agglutinine titles for the homologous serovar, expressed on base 10 logarithm, were at least 1.19.

O emprego do hamsters (Mesocricetus auratus) como modelo biológico experimental para a reprodução da condição de portador renal de leptospiras foi investigado em machos e fêmeas jovens com 80 a 120 g de peso vivo. Os animais foram experimentalmente infectados com estirpe patogênica do sorovar Pomona caracterizada por provocar a morte por leptospirose entre o quinto e o décimo dia pós-infecção. No segundo dia pós-infecção os animais foram tratados com estolato de eritromicina, nas concentrações de 10, 20, 40 e 80 mg/kg de peso vivo. Aos 30 dias, da infecção experimental os sobreviventes foram anestesiados com isofluorano e procedeu-se a colheita de sangue para a determinação dos indicadores da função hepática e renal (Proteínas totais, Albumina, Uréia, Creatinina, Fosfatase Alcalina, Alanina Aminotransferase, Aspartato Aminotransferase, Bilirrubinas Indireta, Bilirrubinas Direta e Bilirrubinas Totais), bem como o titulo de aglutininas pela prova de soro aglutinação microscópica. A seguir, com o aprofundamento da anestesia, os animais foram submetidos a eutanásia e necropsiados para a colheita de tecido renal e hepático destinados aos exames histopatológicos pelas colorações de Hematoxilina - Eosina e Warthin- Starry, bem como do isolamento de leptospiras por cultivo em meio de Fletcher. Houve controles do inóculo infeccioso, do tratamento com antibiótico e do sistema de manejo adotado. O número de DL 50 efetivamente empregadas no inóculo infeccioso foi de 7,11. No grupo controle do antibiótico foi constatado elevação do nível de fosfatase alcalina e degeneração vacuolares dos hepatócitos para as concentrações de 40 a 80 mg de antibiótico. Os portadores renais de leptospira foram obtidos entre os animais tratados com 40 ou 80 mg de estolato de eritromicina, independentemente do sexo; estes animais apresentaram elevação dos níveis séricos de creatinina e proteínas totais já as determinações de albumina, uréia, alanina aminotrasferase, aspartato aminotransferase, bilirrubinas direta, bilirrubinas indiretas e totais foram iguais as encontradas em animais não infectados por leptospiras e não tratados com antibióticos. As alterações histológicas encontradas nos animais portadores de leptospiras foram degeneração vacuolar em hepatócitos, sangue no espaço porta, congestão glomerular. Nos animais induzidos a condição de portadores renais de leptospiras os títulos de anticorpos aglutinantes, para o sorovar homólogo ao da infecção, expressos em logaritmo de base, 10 foram iguais ou superiores a 1,19. / The use of hamsters (Mesocricetus auratus) as experimental model for the reproduction of leptospires kidney carrier condition was investigated on youngs males and females with 80 to 120 g of living weight. The animals were experimentally infected with pathogenic strain of serovar Pomona able of causing the death by leptospirosis between the fifth and the tenth post-infection day. On the second day post-infection the animals were treated with erythromycin estolate at the concentrations of 10, 20, 40 and 80 mg/ kg of living weight. At the 30th day of experimental infection the survivors were anesthetized with isoflurane and blood sample were collected for the determination of kidney and liver functions (Total proteins, Albumin, Urea, Creatinine, Alkaline Phosphatase, Alanine Aminotransferase, Aspartate Aminotransferase, Indirect Bilirubins, Direct Bilirubins and Total Bilirubins), and the of agglutinine title by the microscopic agglutination test (MAT). The animals were killed whit the reinforcement of the anesthesis, and necropsied for the collection of kidney and liver sample for histopathologic tests by staining of Hematoxylin-Eosina and Warthin-Starry, such as the isolation of leptospiras by cultivation into Fletcher´s medium. There were controls of the infecctious inoculum, antibiotic treatment and of the management system adopted. The number of DL 50 effectively applied in the infectious inoculum was 7,11. The antibiotic controls presented elevation of the alkaline phosphatase level and vacuolar degeneration of hepatocytes at the concentrations of 40 to 80 mg of antibiotic. The leptospire\'s kidney carriers were obtained in the animals treated with 40 or 80 mg of Erythromycin Estolate, regardless of the sex; these animals showed increase in creatinine\'s and total protein serum levels but of albumin, urea, alanine aminotransferase, aspartate aminotransferase, direct bilirubins, indirect bilirubins and total bilirubins were the same as found in animals not infected by leptospires and not treated with the antibiotic. The histological changes found in the animals induced as leptospires carrier were vacuolar degeneration in hepatocytes, blood in the portal tract, and glomerular congestion. The agglutinine titles for the homologous serovar, expressed on base 10 logarithm, were at least 1.19.

The mammalian embryo is encased in a glycoproteinaceous covering, the zona pellucida (ZP/zona) during preimplantation development. Prior to implantation into the recipient maternal endometrium, the blastocyst has to hatch out of this zona. This is a critical and an important event for the successful establishment of pregnancy. Hatching in mammals is characterised by the expansion of the blastocyst, followed by the nicking of the zona and extrusion of the blastocyst by repeated contraction-expansion cycles, thereby leaving the empty zona behind. In species such as the mouse, cow and primates, the empty zona is left behind in the uterine lumen. However, in the hamsters, the features associated with hatching are characteristic for this species. Firstly, the blastocyst remains predominantly in a deflated state. Secondly, the zona undergoes focal rupture which is followed by the complete dissolution of the zona. Third, trophectodermal projections (TEPs) present in the blastocysts, aid the hatching of the blastocyst. Hence, this study was aimed to identify the molecular players involved in hamster blastocyst hatching and to study their embryo-endometrial expression. Earlier work in the laboratory has demonstrated the involvement of cysteine protease-like factors in hamster blastocyst hatching (Mishra and Seshagiri, 2000a). Broad spectrum cysteine protease inhibitors, E-64 and PHMB, completely blocked the hatching of blastocysts. To identify the class of cysteine proteases involved in this phenomenon, class-specific inhibitors were used in this study. Calpain and caspase inhibitors, calpastatin and Z-VAD- FMK, respectively, did not block hamster blastocyst hatching (Fig 2.2). Cathepsin (cts)-specific inhibitors, cystatin-C and peptidyl diazomethane (PPDM) blocked hatching of embryos in a dose-, time- and embryo-stage dependant manner (Figs 2.5, 2.6, 2.10 and 2.11). Continuous exposure of 1.0 µM cystatin to expanded or deflated blastocysts completely blocked hatching (Fig 2.3Aii, iii), without effecting their viability (Fig 2.3Bii and iii). Deflated blastocysts exposed transiently to 1.0 µM cystatin, for 12 or 6 h failed to hatch, but could overcome the inhibition and exhibited hatching, when transferred to fresh, inhibitor-free medium (Fig 2.6). Effect of the inhibitor was less pronounced in the deflated blastocysts when compared to expanded blastocysts (Figs 2.5 and 2.6). The viability of the cystatin-treated embryos was not affected as assessed by vital-dye staining and also their ability to attach and exhibit trophoblast (TB) proliferation on serum-coated dishes was not compromised. The area of the TB outgrowth of cystatin-treated embryos was similar to that of the untreated embryos (Fig 2.9). The inhibitory effect of PPDM, an irreversible inhibitor of cts, on blastocyst hatching was demonstrated. Expanded and deflated blastocysts exhibited a dose-dependent inhibition in hatching, following the exposure to 0.5 and 1.0 µM of PPDM (Figs 2.10A and 2.11A). When treated with the inhibitor for 6 or 12 h, there was a transient inhibition in hatching, as blastocysts could overcome the inhibition and exhibit hatching following transfer to inhibitor-free, fresh medium. Inhibitor-treated hatched blastocysts, when transferred to serum-coated dishes, attached and exhibited TB outgrowth, similar to untreated embryos (Figs 2.13 and 2.14). A PPDM-interacting protease was localised to the cytoplasm of the embryonic cells in the hamster blastocyst, suggesting that the embryo is the source of the zona lysin. Two forms of the enzyme, a probable variant zymogen of molecular mass 65 k and an active form of molecular mass 32 k were detected in the blastocysts (Fig 2.15). In vitro susceptibility of hamster zona to cathepsins is significantly different from that of other species zonae such as the mouse, rat, monkey and human zonae (Table 2.2). All these lines of evidence unequivocally demonstrate the involvement of cathepsins in hamster blastocyst hatching, which is in sharp contrast to what is observed in the mouse, where serine proteases such as strypsin/hepsin, ISP-1 and -2 are reported to play an important role in blastocyst hatching. However, since extensive inhibitor studies were not performed using embryos from other species, it is possible that cysteine proteases maybe involved in the hatching of blastocysts from other species. Having shown the role of cathepsins in hamster zona dissolution, expression of the cathepsins in preimplantation embryos was investigated. Hamster specific cts–L, -B and –P were amplified from day 14 placenta using mouse primers and the amplicons were found to be highly homologous to the cts of other species (Fig 3.2). Hamster and mouse preimplantation embryos i.e., 8-cell, morula and blastocyst were found to express cts–L, -B and –P transcripts (Figs 3.6 and 3.10). Cts-P, present only in the TBs of the placenta (Fig 3.4), for the first time, was also shown to be present in the preimplantation embryos. The immunoreactive cts-L and -P proteins were detected in blastomeres of 8-cell embryo, in the inner cell mass (ICM) and trophectoderm (TE) of the blastocyst (Figs 3.7 and 3.8). These cathepsins could probably correspond to the PPDM- interacting enzymes of molecular mass 32 and 65 kDa, described above. (fig) Fig 5.1. Overview of the expression and the role of embryo-endometrial cathepsins in blastocyst hatching in the golden hamster. Cathepsins ( ) produced by the inner cell mass ( ) or the trophectoderm ( ) of the blastocyst or the endometrial cells ( ) act on the zona matrix ( ), bringing about its lysis. The cathepsins are secreted into the peri-vitelline space or are carried by trophectodermal projections (TEPs, yellow projections) to the zona. Also shown are endogenous inhibitors and growth factors that can regulate these cathepsins. A striking observation made in this study was the detection of the immunoreactive signals for cathepsins in the zona matrix of blastocysts. Since hamster blastocysts possess extracellular projections (TEPs), it is possible for these projections to participate in the transport of cathepsins from TE cells to the zona; as the localisation of the proteases to these projections was demonstrated (Fig 3.9). Also, since the actin-based projections are highly undulating structures, they might potentiate the mechanical rupture of the zona during hatching, apart from acting as carriers for the proteases. Hence, during hatching of the hamster blastocyst, cathepsins, expressed in the ICM and the TE, might be secreted transiently into the peri-vitelline space, whereby they can act on the ZP. Alternatively, in the absence of any apoptotic cells in the embryo that can release the cell contents (Fig 3.13), the cathepsins may be deposited by TEPs in specific pockets of the zona matrix, thereby causing focal zona lysis. In vivo, the hatching of the blastocysts is brought about by both embryonic and maternal proteases. Cts–L and -B transcripts were detected in the maternal endometrium during different stages of the reproductive cycle and early pregnancy (Fig 4.1 and 4.3). Immunoreactive cts-L protein was detected in the uterine luminal epithelium and the stromal cells (Fig 4.5). In the uterus, the PPDM-interacting 32 kDa form was in abundance compared to the 65 kDa form (Fig 4.6). Hence, uterine cathepsins might play a major role in the remodelling of the extracellular matrix during estrous cycle and pregnancy. However, the role of these cathepsins in causing zona dissolution during blastocyst hatching, along with embryonic proteases cannot be ruled out. Reports of recurrent miscarriages in women with low serum cystatin levels imply a role for cysteine proteases in early pregnancy events like blastocyst development, hatching and implantation. Hence, these studies, described in the thesis, could form a basis to investigate the role of cathepsins in early human development. Taken together, the results demonstrate the involvement of embryo-derived cathepsins in hamster blastocyst hatching. These cathepsins may be secreted into the peri-vitelline space or transported to the zona matrix by TEPs (Fig 5.1). Additionally, in vivo, endometrial cathepsins might aid the embryonic zona lysins in the complete zona dissolution. The regulation of these proteases by growth factors, cytokines and their specific inhibitors needs to be explored. (For figure pl see the original document)

Not available / text

En raison de ses formes peu biodisponibles retrouvées dans la nature, le silicium n'a reçu que très peu d'intérêt dans le domaine biologique. Or, il est clairement démontré que le silicium peut avoir un rôle important dans la prévention de l'athérosclérose. L'entreprise Phyco-Biotech, productrice de spiruline, s'est intéressée à ce problème. Les propriétés nutritionnelles exceptionnelles de la spiruline, ainsi que ses effets bénéfiques sur certaines pathologies ne sont plus à démontrer. Grâce à un procédé breveté, Phyco-Biotech a réussi à produire une spiruline enrichie en silicium, permettant au silicium d'avoir certainement une meilleure biodisponibilité chez l'homme. L'objectif de ce travail a été d'étudier les effets d'une supplémentation en spiruline enrichie en silicium, en mode préventif, sur un modèle animal d'athérosclérose nutritionnellement induite. Dans une étude de toxicologie chez le rat, constituant la première partie de ce travail, nous avons montré que la spiruline enrichie en silicium n'est pas toxique à la dose utilisée. Dans la seconde partie, nous avons mis en évidence un effet préventif de la spiruline enrichie en silicium sur les multiples composantes de la pathologie athéromateuse et sur les désordres nutritionnels liés à la consommation d'un régime riche en graisses saturées. A la suite de ce travail, nous avons proposé des hypothèses de mécanismes d'action. Ces mécanismes pourraient impliquer une modulation de la voie NF-κB ; ou bien agir sur le remodelage artériel et notamment sur les processus cellulaires impliqués dans les propriétés contractiles de l'artère. La compréhension totale du mécanisme d'action de cette spiruline enrichie en silicium nécessite cependant plus d'investigations. / Due to low bioavailability forms of silicon found in the nature, it has received little interest in the biological field. However, it was clearly demonstrated that silicon may have an important role in prevention of atherosclerosis.The company Phyco-Biotech, spirulina producer, was interested in this problem. The exceptional nutritional properties of spirulina and its beneficial effects on some diseases are well established. Through a patented process, Phyco-Biotech has successfully produced silicon enriched spirulina, allowing the silicon to have a better bioavailability in humans.The objective of this work was to study the effects of a supplementation with silicon enriched spirulina, in an animal model of nutritionally induced atherosclerosis.The first part of this study has shown, using a study in rats, that silicon enriched spirulina was not toxic at the dose used.In the second part, silicon enriched spirulina has shown a preventive effect on risk factors for atherosclerotic disease and disorders related to the consumption of a high fat diet.This work allowed to propose hypothesis of mechanisms of action. Indeed, these mechanisms could involve modulation of the NF-kB pathway, or act on the arterial remodeling or on cellular pathways involved in the contractile properties of the artery. More investigations are needed to determine the complete mechanism of action of this silicon enriched spirulina.

Este trabalho teve por objetivo avaliar as alterações respiratórias e neurológicas resultantes da infecção por via intranasal das diferentes estirpes nacionais do herpesvírus equino tipo 1 (EHV-1) em hamsters comparando sua susceptibilidade com estudos sobre infecção do EHV-1 em camundongos e equinos. Para isso, hamsters sírios machos, três semanas de idade, foram infectados por via intranasal com as estirpes do EHV-1 obtidas a partir de fetos abortados e potro neonato infectados (A4/72, A9/92, A3/97 e Iso/72). Os animais foram pesados e examinados diariamente em busca de sinais clínicos e neurológicos. Conforme os sintomas neurológicos apareceram, grupos de cinco hamsters foram eutanasiados por overdose de isoflurano na primeira etapa do projeto, e, de cetamina/xilazina por via intraperitoneal na segunda etapa. Na necropsia, sistema nervoso central (SNC), pulmão, fígado, baço e timo foram coletados para isolamento viral em cultura de células E-dermal e para análise histopatológica. Na segunda etapa do projeto, a lavagem broncoalveolar (LBA) com 3 ml de PBS por hamsters foi realizada para determinar a resposta inflamatória pulmonar através da contagem total e diferencial de leucócitos. Similar aos experimentos com camundongos, hamsters desafiados com as estirpes virais A4/72 e A9/92 apresentaram manifestações clínicas severas no terceiro dia pós-inoculação (dpi), tais como perda de peso aguda, dispnéia, desidratação, decúbito e morte. Observou-se também sinais neurológicos como hiperexcitabilidade, paralisia, espasmos, perda de propriocepção, andar em círculos e convulsões. Ao contrário dos camundongos, que não desenvolveram a doença; hamsters inoculados com as estirpes virais A3/97 e Iso/72 manifestaram sintomas respiratórios e neurológicos agudos entre quarto e quinto dpi, sendo as alterações respiratórias as mais evidentes, principalmente hemoptise e conjuntivite. O isolamento do vírus a partir do SNC foi positivo em todos os animais infectados; no entanto, todas as amostras de pulmões foram positivas apenas nos grupos infectado pelas estirpes virais A9/92 e A4/72. Todas as estirpes do EHV-1 foram isoladas a partir do baço, no entanto, a partir do timo foram isoladas apenas as estirpes A9/92 e A4/72, e, de fígado somente a estirpe viral Iso/72 não foi isolada. No LBA, a contagem total de leucócitos não demonstrou diferenças de valores significativas entre os grupos experimentais, sendo apenas evidente a presença de eritrócitos, macrófagos e neutrófilos na maioria dos esfregaços dos hamsters infectados. Porém, na contagem diferencial de leucócitos observou-se um aumento significativo de neutrófilo com consequente diminuição significativa de macrófagos nos hamsters infectados pelas estirpes virais A3/97, Iso/72 e A4/72 quando comparados ao grupo controle. Os hamsters infectados pela estirpe viral A9/92 mantiveram valores próximos aos do grupo controle. Ao avaliar microscopicamente o SNC, fígado e pulmão dos hamsters infectados foi observado infiltrado inflamatório, necrose e manguito perivascular em SNC; leucocitose e necrose no parênquima hepático com discreta pericolangite e proliferação de ducto biliar; e em pulmão observou inflamação, necrose, edema e hemorragia em bronquíolos e alvéolos. Concluindo, os resultados apontaram o hamster como a espécie mais susceptível à infecção pelo EHV-1 servindo como um modelo experimental complementar para estudos de doenças respiratória e neurológica provocadas por este agente em equinos / This study aimed to evaluate the respiratory and neurological disorders resulting from intranasal infection with different national strains of equine herpesvirus type 1 (EHV-1) in hamsters comparing their susceptibility to studies about EHV-1 infection in mice and horses. Therefore, male Syrian hamsters, three weeks of age, were infected via intransal with EHV-1 strains obtained from aborted fetuses and neonatal foal infected (A4/72, A9/92, A3/97 and Iso/72). The animals were weighed and examined daily for clinical search and neurological signs. As neurological symptoms appeared five hamsters groups were euthanized by isoflurane overdose in the first stage of the project, and ketamine/xylazine via intraperitoneal in the second stage. At necropsy, central nervous system (CNS), lung, liver, spleen and thymus were collected for virus isolation in E-dermal cell culture and histopathological analysis. In the second stage of the project, bronchoalveolar lavage (BAL) with 3 ml of PBS per hamsters was performed to determine the pulmonary inflammatory response through the total and differential leukocyte count. Similar to the experiments with mice, hamsters challenged with viral strains A4/72 and A9/92 had severe clinical manifestations in the third day post-inoculation (dpi), such as acute weight loss, dyspnea, dehydration, recumbency, and death. It was also observed neurological signs such as hyperexcitability, paralysis, spasms, loss of proprioception, circling and convulsions. Unlike mice, which did not develop disease; hamsters inoculated with viral strains A3/97 and Iso/72 showed acute respiratory and neurological symptoms between fourth and fifth dpi, and the most evident respiratory distress was hemoptysis and conjunctivitis. The virus isolation from CNS was positive in all infected animals; however, all lung samples were positive only in groups infected by A9/92 and A4/72 viral strains. All EHV-1 strains were recovered from spleen, however, only A9/92 and A4/72 viral strains were recovered from thymus, but only Iso/72 viral strain was not recovered from liver. BAL total leukocyte count showed no significant differences in values between the experimental groups but the presence of erythrocytes, macrophages and neutrophils were evident in most smears of infected hamsters. However, in the leukocyte differential count it was observed a significant increase in neutrophils with consequent significant reduction of macrophages in hamsters infected by viral strains A3/97, Iso/72 and A9/92 when compared with the control group. Hamsters infected with the A9/92 viral strain maintained values similar to the control group. When evaluating microscopically the CNS, liver and lungs of infected hamsters it was observed inflammatory infiltrate, necrosis and perivascular cuff in CNS; leukocytosis and necrosis in the liver parenchyma with discrete pericholangitis and bile duct proliferation; and inflammation, necrosis, edema and hemorrhage in the bronchioles and alveoli. Concluding, the results showed the hamster as the most susceptible species to EHV-1 infection serving as a complementary experimental model for studies of respiratory and neurological diseases caused by this agent in horses

A produção de proteínas recombinantes, principalmente para uso farmacêutico, tem sido intensamente estudada, juntamente com suas propriedades físico-químicas, o que possibilita uma melhor escolha da técnica de purificação, e assim, evitar perdas no rendimento e custos elevados. A glicocerebrosidase (GCR) é uma enzima lisossomal, e sua deficiência ocasiona um distúrbio autossômico recessivo denominado doença de Gaucher. Atualmente o tratamento para essa patologia é por meio da Terapia de Reposição Enzimática (TRE), a qual tem sido realizada com grande êxito. Tendo em vista fornecer dados que possam auxiliar na redução do número de etapas cromatográficas no processo de downstream, este trabalho teve como objetivo, a purificação da GCR expressa por células de Ovário de Hamster Chinês (CHO), em meio livre de soro fetal bovino (SFB). Para se alcançar tais resultados, realizou-se o cultivo das células CHO-GCR em meio quimicamente definido livre de SFB (CHO-S-SFM II). Foram realizadas três técnicas cromatográficas (troca iônica, interação hidrofóbica e afinidade), com base em suas propriedades físico-químicas. E para o ensaio da atividade enzimática, foi utilizado o substrato fluorogênico 4-Methylumbeliferyl β-D-glucopyranoside (4MU-G). As células CHO-GCR cultivadas em meio CHO-S-SFM II, apresentando viabilidade maior que 95%, e produção da GCR ativa, durante todo o período de cultivo. Os protocolos aplicados para a purificação da GCR, não apresentaram resultados significativos. Com o volume não retido após cromatografia por interação hidrofóbica, se estimou os valores de KM 2,13 e VMAX 0,0295 UFR/h para as constantes cinéticas da GCR. A diálise no processo de purificação mostrou ser uma etapa necessária para a atividade enzimática da GCR. No cultivo das células CHO-GCR para a formação do banco de trabalho, nos meios RPMI 1640 e α-MEM, ambos com a adição de 10% SFB, não houve diferença significativa no crescimento entre eles, e apresentaram 100% de viabilidade durante todo o período de cultivo. Porém, ao analisar de forma isolada a fase exponencial de cada curva, notou-se que às células cultivadas no meio RPMI 1640, apresentaram taxa de crescimento superior, às cultivadas em meio α-MEM. Concluiu-se que a expressão da GCR em meio livre de SFB, proporciona amostras menos complexas, em relação aos meios de cultura que necessitam de suplementação com SFB, o que pode reduzir o número de etapas cromatográficas, melhorando o rendimento e a redução da perda da atividade da GCR. O meio basal RPMI 1640 com a adição de SFB foi uma alternativa satisfatória para o cultivo das células CHO-GCR. Este estudo forneceu dados que podem contribuir para a melhoria do processo de purificação da GCR. Novas pesquisas podem ser desenvolvidas a fim de melhorar o processo de purificação da GCR. / The production of recombinant proteins, mainly for pharmaceutical use, has been intensively studied along with its physico-chemical properties, which allows a better choice of the purification technique, and thus avoid losses in yield and high costs. Glucocerebrosidase (GCR) is a lysosomal enzyme, and its deficiency causes an autosomal recessive disorder called Gaucher\'s disease. Currently the treatment for this pathology is through Enzymatic Replacement Therapy (ERT), which has been successfully performed. In order to provide data that may help reducing the number of chromatographic steps in the downstream process, this work aimed to purify GCR expressed by Chinese Hamster Ovary (CHO) cells in fetal bovine serum free (FBS). To achieve such results, the CHO-GCR cells were cultured in chemically defined Serum-free medium (CHO-S-SFM II). Three chromatographic techniques (ion exchange, hydrophobic interaction and affinity) were performed, based on their physicochemical properties. And for the enzymatic activity assay, the fluorogenic substrate 4-Methylumbeliferyl β-D-glucopyranoside (4MU-G) was used. CHO-GCR cells cultured in CHO-S-SFM II medium, presenting viability greater than 95% and GCR production active, throughout the culture period. The protocols applied for GCR purification did not present significant results. With the volume not retained after chromatography by hydrophobic interaction, KM values of 2.13 and VMAX 0.0295 UFR/h were estimated for GCR kinetic constants. Dialysis in the purification process was shown to be a step necessary for the enzymatic activity of GCR. In the culture of the CHO-GCR cells for the formation of the working bank, in the media RPMI 1640 and α-MEM, both with the addition of 10% FBS, there was no significant growth difference between them, and they showed 100% viability during all the growing period. However, when analyzing in isolation, the exponential phase of each curve, it was observed that the cells grown in RPMI 1640 medium showed higher growth rates, tham grown in α-MEM medium. It was concluded that the expression of GCR in serum-free medium provides less complex samples, relative to the culture media requiring FBS supplementation, which may reduce the number of chromatographic steps, improving yield and loss reduction of GCR activity. The basal medium RPMI 1640 with the addition of FBS was a satisfactory alternative for culturing the CHO-GCR cells. This study provided data that may contribute to the improvement of the GCR purification process. New research can be developed to improve the GCR purification process.

A prolactina humana (hPRL) é um hormônio polipeptídico secretado pela hipófise anterior sob regulação do hipotálamo, envolvido em uma variedade de processos biológicos como o desenvolvimento da glândula mamária e lactação. O produto recombinante é importante no diagnóstico médico e no tratamento de insuficiência da lactação. Este hormônio pode ocorrer sob a forma de proteína não glicosilada (NG-hPRL) e glicosilada (G-hPRL), com pesos moleculares de aproximadamente 23 e 25 kilodalton (kDa), respectivamente; possui um único sítio de N-glicosilação localizado na asparagina (Asn) posição 31, que é parcialmente ocupado, representando assim um modelo particularmente interessante de glicosilação. A atividade biológica da G-hPRL é muito menor comparada à NG-hPRL (~4 vezes) e sua função fisiológica ainda não é bem definida: a porção de carboidrato parece ter um importante papel na biossíntese, secreção, atividade biológica, e sobrevivência plasmática do hormônio. O objetivo principal desse trabalho foi comparar as estruturas dos N-glicanos presentes na prolactina glicosilada hipofisária (G-hPRL-NHPP) com a recombinante. Para obter a G-hPRL recombinante foi realizada uma produção em escala laboratorial a partir de células de ovário de hamster chinês (CHO) geneticamente modificadas e adaptadas ao crescimento em suspensão. Foi adicionada, ao meio de cultura cicloheximida (CHX), cujo efeito principal foi aumentar a relação G-hPRL/NGhPRL que passou de 5% para 38%, facilitando assim a purificação da G-hPRL. A G-hPRL foi purificada em duas etapas, uma troca catiônica seguida de purificação por cromatografia liquida de alta eficiência de fase reversa (RP-HPLC) que se demonstrou eficiente na separação das duas isoformas de hPRL. A G-hPRL recombinante IPEN foi assim analisada por diversas técnicas confirmando a sua pureza e atividade biológica, incluindo comparações com outras amostras de referências de origem hipofisária adquirida junto ao National Hormone & Peptide Program (NHPP-E.U.A.) . Foi realizada também a determinação inédita de Nglicanos presentes na G-hPRL produzida por células CHO e na G-hPRL nativa, produzida pela hipófise humana, possibilitando comparar as duas estruturas de carboidratos e alcançando assim uma das principais metas desse projeto. Entre as principais diferenças encontradas nas estruturas dos dois N-glicanos, destacam-se a baixa quantidade de ácido siálico (NeuAc), a alta porcentagem de glicanos sulfatos (74,0%) e com fucose (Fuc) (93,3%) presentes na amostra hipofisária e a tendência da preparação recombinante de apresentar glicanos com maior peso molecular e com uma menor variação nas isoformas. / Human prolactin (hPRL) is a polypeptide hormone secreted by the anterior pituitary under the regulation of the hypothalamus, involved in a variety of biological processes such as mammary gland development and lactation. The recombinant product is important in medical diagnosis and treatment of failure of lactation. This hormone may occur in the form of non-glycosylated protein (NGhPRL) and glycosylated (G-hPRL) with molecular weights of approximately 23 and 25 kilodalton (kDa), respectively; has a single N-glycosylation site located at asparagine (Asn) position 31, which is partially occupied, thus being a particularly interesting model of glycosylation. The biological activity of G-hPRL is lower compared to NG-hPRL (~4 times) and its physiological function is not well defined: the portion of carbohydrate appears to have an important role in the hormone biosynthesis, secretion, biological activity, and plasma survival of the hormone. The main objective of this study was to compare the structures of N-glycans present in glycosylated pituitary prolactin (G-hPRL-NHPP) with those present in the recombinant. To obtain the recombinant G-hPRL the production was performed in laboratory scale from Chinese hamster ovary cells (CHO), genetically modified and adapted to growth in suspension. Cycloheximide (CHX), whose main effect was to increase the ratio G-hPRL/NG-hPRL from 5% to 38% was added to the culture medium, thereby facilitating the purification of G-hPRL. The G-hPRL was purified in two steps, a cation exchanger followed by a purification by reversed-phase high performance liquid chromatography (RP-HPLC) which demonstrated the efficient separation of the two isoforms of hPRL. Recombinant G-hPRL-IPEN was well characterized by several techniques confirming its purity and biological activity, including comparisons with other reference preparation of pituitary origin purchased from the \"National Hormone & Peptide Program (NHPPU. S.)\". The composition of N-glycans present in the G-hPRL, produced by CHO cells, and that of native G-hPRL, produced by the human pituitary gland, were also determined for the first time, allowing the two structures of carbohydrates to be compared and thus, achieving one of the main goals of this project. Among the main differences in N-glycan structures, we highlight the low presence of sialic acid (NeuAc) and the high percentage of sulfated glycans (74.0%) and of fucose (Fuc) (93.3%) in the pituitary sample and the tendency of the recombinant preparation to present glycans with higher molecular weight and less isoforms variation.