RECK(reversion-inducing cysteine-rich protein with Kazal motifs) encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeat, four N-glycosylation sites and three Kazal-like domains. RECK functions as a tumor suppressor gene which may inhibit the release and activation of MMP-2 and MMP-9. Previous studies indicated that RECK-mediated suppression of tumor cell invasion is regulated by glycosylation of RECK in human tumor cell lines. However, the anti-cancer action of other functional domains of RECK have not been studied. In the study, We investigated the effects of different functional domains of RECK protein on the invasion of tumor cell lines and on the activation of matrix metalloproteinase. We constructed bacterial expression vector and secretory mammalian expression vector which could produce full-length, Kazal-like motifs 1~3, Kazal-like motifs 2~3 and CKM5 polypeptides. Recombinant proteins were purified and used for treatment of human lung cancer cell lines. We found that treatment of K23 and RECK recombinant proteins resulted in suppression of invasive ability and MMP activity. Moreover,K23 and RECK proteins were found to inhibit the secretion of matrix metallo- protease-9 (MMP-9). K23 also formed a complex with MMP-9 and inhibited its proteolytic activity noncompetitively. Experimental metastasis assay revealed that there were fewer tumor nodule formation in the lungs injected with A549 cells stably expressing K23 than control vector. Thus, these findings indicate that the K23 domain of RECK functions as an inhibitor of tumor invasion and metastasis.

A pancreatite crônica é uma desordem complexa, na qual a interação entre fatores ambientais e genéticos resulta na enfermidade. O presente estudo incluiu 148 pacientes com diagnóstico de pancreatite crônica, 110 etilistas crônicos e 297 controles sadios com o objetivo de investigar a frequência de tabagismo e das mutações N34S e P55S do gene SPINK1 e R254W do gene CTRC nesta população. Foi aplicado questionário presencial e realizada reação de sequenciamento para a pesquisa das mutações genéticas, após assinatura do Termo de Consentimento Livre e Esclarecido. Os portadores de pancreatite crônica possuíam etiologia alcoólica em 74% das vezes e idiopática em 26%. A pancreatite alcoólica apresentou-se de maneira distinta da pancreatite crônica idiopática, sendo que o primeiro grupo é composto por maior prevalência do gênero masculino (88,18% versus 34,21%), por maior média de idade (55,64 anos versus 45,20 anos), menor frequência de caucasianos (63,89% versus 84,21%), menor escolaridade (23,30% concluíram ensino médio ou superior versus 57,89%) e maior frequência de repercussões da doença, como diarréia (54,21% versus 24,24%), emagrecimento (56,07% versus 24,24%), diabete melito (57,94% versus 36,36%) e ocorrência de pseudocistos pancreáticos (31,78% versus 12,12%), repercussões estas que não foram acompanhadas de maior frequência de alterações morfológicas, como calcificações pancreáticas ou dilatação do ducto pancreático principal. A frequência de tabagismo foi significativamente maior em pacientes com pancreatite crônica alcoólica do que em etilistas sem pancreatite crônica, podendo ser considerado cofator de risco para o desenvolvimento da pancreatite crônica entre alcoolistas (p = 0,002); a frequência da mutação N34S do gene SPINK1 em pacientes com pancreatite crônica foi de 3,38%, maior do que a frequência de 0,49% encontrada nos grupos controle (p = 0,016); a frequência de 2,03% da mutação P55S do gene SPINK1 e a frequência de 0,67% da mutação R254W do gene CTRC, encontradas nos pacientes com pancreatite crônica, não diferiram estatisticamente quando comparadas às frequências, de 0,49% de ambas mutações, encontradas nos grupos controle. (p = 0,120 e 0,751). Pela investigação da associação de tabagismo e da mutação N34S do gene SPINK1 com as características clínicas e morfológicas da pancreatite crônica, verificou-se que a mutação N34S não se associou a maior gravidade da apresentação clínica ou morfológica da pancreatite crônica; no entanto o tabagismo associou-se a maior frequência de diabete melito entre os portadores de pancreatite crônica. Concluiuse que o tabagismo e a mutação N34S do gene SPINK1 podem ser considerados cofatores de risco para o desenvolvimento da pancreatite crônica / Chronic pancreatitis is a complex disorder in which the interaction between environmental and genetic factors results in the disease. This study included 148 patients with chronic pancreatitis, 110 chronic alcoholics and 297 healthy controls in order to investigate the frequency of smoking and N34S and P55S mutation of SPINK1 gene and R254W of CTRC gene in this population. A questionnaire was applied and gene sequencing was done, after having the Informed Consent Statement. Those with chronic pancreatitis had alcoholic etiology in 74% of cases and idiopathic in 26%. Alcoholic pancreatitis presented in a distinct way of idiopathic chronic pancreatitis. The first group is composed of a higher prevalence of males (88.18% versus 34.21%), by higher mean age (55.64 years versus 45.20 years), lower frequency of Caucasians (63.89% versus 84.21%), lower education (23.30% completed secondary or higher education versus 57.89%) and worst impact from the disease such as diarrhea (54.21% versus 24.24%), weight loss (56.07% versus 24.24%), diabetes mellitus (57.94% versus 36.36%) and occurrence of pancreatic pseudocysts (31.78% versus 12 , 12%). These effects were not accompanied by increased frequency of morphological changes, such as pancreatic calcifications or dilation of the main pancreatic duct. The frequency of smoking was significantly higher in patients with alcoholic pancreatitis than in alcoholics without chronic pancreatitis, therefore tabagism may be considered as a cofactor for the development of chronic pancreatitis among alcoholics (p = 0.002); the frequency of N34S mutation of SPINK1 gene in patients with chronic pancreatitis was 3.38%, higher than the rate of 0.49% found in the control groups (p = 0.016); the frequency of 2.03% of the P55S mutation of SPINK1 gene and the frequency of 0.67% of the CTRC gene R254W mutation found in patients with chronic pancreatitis were not statistically different when compared to the frequencies of 0.49% of both mutations, found in the control groups. (p = 0.120 and 0.751) For the investigation of the association of smoking and N34S mutation of SPINK1 gene with the clinical and morphological features of chronic pancreatitis, it was noticed that the N34S mutation did not determine a greatest severity in the presentation of chronic pancreatitis, however smoking was associated with a higher frequency of diabetes mellitus in patients with chronic pancreatitis. It was concluded that smoking and the N34S mutation of SPINK1 gene are positively correlated with chronic pancreatitis

A pancreatite crônica é uma desordem complexa, na qual a interação entre fatores ambientais e genéticos resulta na enfermidade. O presente estudo incluiu 148 pacientes com diagnóstico de pancreatite crônica, 110 etilistas crônicos e 297 controles sadios com o objetivo de investigar a frequência de tabagismo e das mutações N34S e P55S do gene SPINK1 e R254W do gene CTRC nesta população. Foi aplicado questionário presencial e realizada reação de sequenciamento para a pesquisa das mutações genéticas, após assinatura do Termo de Consentimento Livre e Esclarecido. Os portadores de pancreatite crônica possuíam etiologia alcoólica em 74% das vezes e idiopática em 26%. A pancreatite alcoólica apresentou-se de maneira distinta da pancreatite crônica idiopática, sendo que o primeiro grupo é composto por maior prevalência do gênero masculino (88,18% versus 34,21%), por maior média de idade (55,64 anos versus 45,20 anos), menor frequência de caucasianos (63,89% versus 84,21%), menor escolaridade (23,30% concluíram ensino médio ou superior versus 57,89%) e maior frequência de repercussões da doença, como diarréia (54,21% versus 24,24%), emagrecimento (56,07% versus 24,24%), diabete melito (57,94% versus 36,36%) e ocorrência de pseudocistos pancreáticos (31,78% versus 12,12%), repercussões estas que não foram acompanhadas de maior frequência de alterações morfológicas, como calcificações pancreáticas ou dilatação do ducto pancreático principal. A frequência de tabagismo foi significativamente maior em pacientes com pancreatite crônica alcoólica do que em etilistas sem pancreatite crônica, podendo ser considerado cofator de risco para o desenvolvimento da pancreatite crônica entre alcoolistas (p = 0,002); a frequência da mutação N34S do gene SPINK1 em pacientes com pancreatite crônica foi de 3,38%, maior do que a frequência de 0,49% encontrada nos grupos controle (p = 0,016); a frequência de 2,03% da mutação P55S do gene SPINK1 e a frequência de 0,67% da mutação R254W do gene CTRC, encontradas nos pacientes com pancreatite crônica, não diferiram estatisticamente quando comparadas às frequências, de 0,49% de ambas mutações, encontradas nos grupos controle. (p = 0,120 e 0,751). Pela investigação da associação de tabagismo e da mutação N34S do gene SPINK1 com as características clínicas e morfológicas da pancreatite crônica, verificou-se que a mutação N34S não se associou a maior gravidade da apresentação clínica ou morfológica da pancreatite crônica; no entanto o tabagismo associou-se a maior frequência de diabete melito entre os portadores de pancreatite crônica. Concluiuse que o tabagismo e a mutação N34S do gene SPINK1 podem ser considerados cofatores de risco para o desenvolvimento da pancreatite crônica / Chronic pancreatitis is a complex disorder in which the interaction between environmental and genetic factors results in the disease. This study included 148 patients with chronic pancreatitis, 110 chronic alcoholics and 297 healthy controls in order to investigate the frequency of smoking and N34S and P55S mutation of SPINK1 gene and R254W of CTRC gene in this population. A questionnaire was applied and gene sequencing was done, after having the Informed Consent Statement. Those with chronic pancreatitis had alcoholic etiology in 74% of cases and idiopathic in 26%. Alcoholic pancreatitis presented in a distinct way of idiopathic chronic pancreatitis. The first group is composed of a higher prevalence of males (88.18% versus 34.21%), by higher mean age (55.64 years versus 45.20 years), lower frequency of Caucasians (63.89% versus 84.21%), lower education (23.30% completed secondary or higher education versus 57.89%) and worst impact from the disease such as diarrhea (54.21% versus 24.24%), weight loss (56.07% versus 24.24%), diabetes mellitus (57.94% versus 36.36%) and occurrence of pancreatic pseudocysts (31.78% versus 12 , 12%). These effects were not accompanied by increased frequency of morphological changes, such as pancreatic calcifications or dilation of the main pancreatic duct. The frequency of smoking was significantly higher in patients with alcoholic pancreatitis than in alcoholics without chronic pancreatitis, therefore tabagism may be considered as a cofactor for the development of chronic pancreatitis among alcoholics (p = 0.002); the frequency of N34S mutation of SPINK1 gene in patients with chronic pancreatitis was 3.38%, higher than the rate of 0.49% found in the control groups (p = 0.016); the frequency of 2.03% of the P55S mutation of SPINK1 gene and the frequency of 0.67% of the CTRC gene R254W mutation found in patients with chronic pancreatitis were not statistically different when compared to the frequencies of 0.49% of both mutations, found in the control groups. (p = 0.120 and 0.751) For the investigation of the association of smoking and N34S mutation of SPINK1 gene with the clinical and morphological features of chronic pancreatitis, it was noticed that the N34S mutation did not determine a greatest severity in the presentation of chronic pancreatitis, however smoking was associated with a higher frequency of diabetes mellitus in patients with chronic pancreatitis. It was concluded that smoking and the N34S mutation of SPINK1 gene are positively correlated with chronic pancreatitis

Master of Science / Department of Entomology / Marcelo Ramalho-Ortigao / Sand flies (Diptera:Psychodidae) are vectors of parasites of the genus Leishmania transmitted to suitable vertebrate host during blood feeding. For blood feeding arthropods, including sand flies, blood meal digestion requires the secretion of inhibitory molecules, such as Kazal-type serine proteinase inhibitors that are involved in preventing the blood from coagulating within the mouthparts and the midgut. Previous studies have identified such molecules in mosquitoes, ticks, and triatomine bugs. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). We are interested in the role of these proteins as inhibitors of coagulation cascades, in addition to their potential effects on blood digestion in P. papatasi. Ppkzl1 is similar to thrombin and trypsin inhibitors in triatomines and mosquitoes and Ppkzl2 is similar to Kazal-type inhibitors in mosquitoes with unknown function. Analyses of expression profiles indicated that although both transcripts are expressed prior to blood feeding in the midgut of P. papatasi they are tightly regulated by the blood meal. Reverse genetics studies using RNAi-targeted knockdown of PpKzl1 and PpKzl2 by dsRNA injection did not result in a detectable effect on mRNA expression levels. Thus, we expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess activity against various serine proteinases. Recombinant PpKzl2 inhibited chymotrypsin at nanomolar levels and also inhibited thrombin and trypsin at micromolar levels, suggesting that native PpKzl2 is an active serine proteinase inhibitor and may regulate digestive enzymes and thrombin in the midgut. Leishmania development within the sand fly midgut is faced with several barriers that can severely impact the parasites. For transmission to occur, parasites must be able to overcome these barriers including digestive proteinases, escape from the peritrophic matrix, and midgut attachment. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sand fly midgut. Thus, targeting serine proteinase inhibitors may provide a new strategy to prevent transmission of Leishmania.

Hemocytes are important as storage and producers of proteins of the innate immune defence, as well as actors of the cellular immune response. Therefore the hematopoietic process is critical for survival of most invertebrates. In order to search for molecules of importance for hemocyte development in crayfish we investigated proteins in crayfish plasma, which were increased after microbial challenge. As a result we were able to identify, purify and characterize a new invertebrate cytokine named astakine, and could clearly show that this protein is important for hematopoietic development in vivo as well as in an in vitro cell culture system. Astakine contains a prokineticin (PK) domain shown for the first time in an invertebrate, however, unlike the vertebrate PKs, astakine binds to a cell surface F1 ATP synthase β subunit located on the hematopoietic tissue (hpt) cell membranes. Extracellular ATP synthases as receptors have earlier been reported in different vertebrate cells and here we show that extracellular ATP synthase β subunit acts as a receptor for an invertebrate cytokine and is involved in hematopoiesis. We also found two other groups of proteins, which were increased in plasma after microbial challenge and they were further characterized. A great number of different Kazal type proteinase inhibitors were produced by the hemocytes and this type of proteinase inhibitors have variable reactive sites determining the specificity of their inhibition. In crayfish Kazal inhibitors with similar reactive sites were found as a response to specific microorganisms suggesting that the crayfish Kazal proteinase inhibitors may provide enough variability to participate in diverse innate immune reactions against different pathogens. Antimicrobial peptides were synthesized by the hemocytes and were likewise released in high amount upon microbial infection and we have characterized the main group of cystein-rich crustin-like antimicrobial peptides and investigated their tissue distribution and expression pattern.

Reversion-inducing cysteine-rich with Kazal motif (RECK) is a cell surface anchoring protein, which known for the ability to inhibit matrix metalloproteinases (MMPs) and participate in angiogenesis regulation. The inhibition of membrane type-1 matrix metalloproteinase (MT1-MMP), MMP-2, MMP-7 and, MMP-9 by RECK has been demonstrated. Our previous studies show that RECK expression is suppressed by Ras and Her-2/neu oncogene. In addition, oncogenic Ras activates downstream ERK signaling pathway to increase Sp1/HDAC promoter binding affinity which results in reduction of RECK gene transcription and increase of tumor progression and metastasis. From the clinical investigation, RECK expression is down-regulated in a number of cancer types. In breast cancer, RECK expression is associated with the prognosis of the patients. Recently, single nucleotide polymorphisms (SNPs) of RECK promoter have been suggested to be linked with survival rate and prognosis of breast cancer patients. Whether SNP of the RECK promoter has any effect on RECK expression and its clinical significance is still unclear. . In this study, we investigate -402 SNP at RECK promoter and find this SNP directly affects RECK expression through progesterone receptor binding. Additionally, we also address the -402 SNP in the sample collected from patients and analyze its association with clinicopathological parameters to clarify its clinical significance. Our results suggest that RECK SNP may be an valuable prognosis factor for breast cancer.

Το συμπλήρωμα είναι βασικός παράγοντας της φυσικής ανοσίας (innate immunity) και αποτελεί γέφυρα για την ενεργοποίηση της ειδικής ανοσίας (adaptive immunity). Αποτελείται από ένα σύστημα πρωτεϊνών, που συναντώνται ως ανενεργά προένζυμα και ενεργοποιούνται μέσω πρωτεόλυσης πυροδοτώντας έναν καταρράκτη αντιδράσεων. Οι οδοί ενεργοποίησης του συμπληρώματος καταλήγουν στον σχηματισμό ενός λυτικού συμπλόκου (Membrane Attack Complex – MAC) που καταστρέφει τους παθογόνους μικροοργανισμούς. Οι πρωτεΐνες, συστατικά του συμπλόκου, ανήκουν στην οικογένεια MACPF (MAC – Perforin). Σημαντικό ρόλο στη λειτουργία τους παίζει η πρωτεϊνική περιοχή (module) FIMAC (Factor I Membrane Attack Complex). Η περιοχή αυτή υπάρχει στα συστατικά C6 (Complement component 6) και C7 (Complement component 7) του συμπλόκου MAC και φαίνεται να είναι η περιοχή πρόσδεσής τους με την περιοχή C345C του C5b. Επιπλέον, η περιοχή FIMAC υπάρχει και στον παράγοντα Ι του Συμπληρώματος (Complement Factor I, CFI), ο οποίος συμμετέχει στην αποσταθεροποίηση της κομβερτάσης C3/C5, μέσω του καρβοξυτελικού άκρου της FIMAC που φαίνεται να συνδέεται αλλοστερικά με την πρωτεϊνική περιοχή SP (Serine Protease), που αποτελεί την ενεργή του περιοχή. Το μοντέλο, για την ανάλυση της περιοχής FIMAC βασίστηκε στην περιοχή της πρωτεΐνης φολιστατίνη (follistatin, FS) FD (follistatin Domain), η οποία αναλύθηκε πρόσφατα κρυσταλλογραφικά και με την οποία εμφανίζει ομοιότητα στην αλληλουχία της. Η δομή FD αποτελεί ένα υβρίδιο μιας αμινοτελικής περιοχής EGF (Epidermal Growth Factor) και μίας καρβοξυτελικής περιοχής του ωοβλεννοειδούς (ovomucoid) που ομοιάζουν με τις πρωτεϊνικές περιοχές KAZAL και συναντώνται σε πολλούς αναστολείς σερινικών πρωτεασών. Η περιοχή FD περιέχεται, επίσης, στην πρωτεΐνη αγκρίνη (agrin, AGRN) που αποτελεί συστατικό της βασικής μεμβράνης και παίζει σημαντικό ρόλο στην νευρομυϊκή σύναψη. Η μελέτη των πρωτεϊνικών περιοχών FIMAC και KAZAL αναφορικά με την λειτουργία, την δομή και την εξέλιξή τους αποτέλεσε το αντικείμενο της παρούσας εργασίας. Για την εκπόνηση αυτής της μελέτης χρησιμοποιήθηκαν δεδομένα από βάσεις βιολογικών δεδομένων και εργαλεία βιοπληροφορικής ανάλυσης. Πρωτογενές υλικό της μελέτης αποτέλεσαν οι νουκλεοτιδικές και αμινοξικές αλληλουχίες των γονιδίων C6, C7, CFI, AGRN και FS σε όλους τους οργανισμούς που βρέθηκαν (σπονδυλωτά και ασπόνδυλα), και πιο συγκεκριμένα οι αλληλουχίες που αντιστοιχούν στις πρωτεϊνικές περιοχές FIMAC και KAZAL. Οι πρωτοταγείς δομές των FIMAC ( ̴ 78αα) και KAZAL ( ̴ 55αα) διαφέρουν ως προς το μήκος τους, με μερικές εξαιρέσεις που αφορούν περιοχές KAZAL των πρωτεϊνών AGRN και FS (>80αα). Οι δευτεροταγείς δομές των FIMAC και KAZAL παρουσιάζουν μεγάλη ομοιότητα, φέροντας δομές α – έλικας και β – πτυχωτής επιφάνειας στην αλληλουχία τους. Τέλος, τα μοντέλα προσομοίωσης τριτοταγούς δομής και των δύο περιοχών FIMAC και KAZAL οπτικοποιούν τη διαμόρφωση των δομών της α – έλικας και των β – πτυχωτών επιφανειών στον χώρο. Αξιοσημείωτη είναι η παρουσία σημαντικού αριθμού κυστεϊνικών καταλοίπων στις αλληλουχίες των περιοχών FIMAC (8 – 10 Cys), με μεγαλύτερη συγκέντρωση στο αμινοτελικό άκρο, και KAZAL (4 – 6 Cys) με ομοιόμορφη κατανομή. Εξελικτικά, η εμφάνιση γονιδίων που συμμετέχουν και στις τρεις οδούς ενεργοποίησης του συμπληρώματος και καταλήγουν στη διαμόρφωση του συμπλόκου MAC συνοδεύεται με την εμφάνιση των χονδριχθύων. Πιο συγκεκριμένα, όσον αφορά τα γονίδια του συμπληρώματος που περιλαμβάνουν τις περιοχές FIMAC και KAZAL, ο CFI πρωτοεμφανίζεται στα άγναθα, το C6 στους χονδριχθείς και το C7 στους οστεϊχθείς. Η παρουσία των γονιδίων AGRN και FS έχει πιστοποιηθεί νωρίτερα εξελικτικά στο στάδιο των κεφαλοχορδωτών καθώς και στους πλατυέλμινθες των πρωτοστομίων. Έτσι, φαίνεται ότι η περιοχή KAZAL στις πρωτεΐνες AGRN και FS στα ασπόνδυλα αποτελεί προγονική περιοχή όλων των FIMAC και KAZAL που υπάρχουν σήμερα. Ωστόσο, νέες πρωτεϊνικές περιοχές KAZAL εμφανίστηκαν και αργότερα κατά την εξέλιξη των ειδών. Η συντηρητικότητα και των δύο περιοχών FIMAC και KAZAL στο επίπεδο της γονιδιακής τους κληρονόμησης είναι μεγάλη. Όλα τα intron phases των εξονίων που κωδικοποιούν τις περιοχές KAZAL και FIMAC σε όλα τα γονίδια όπου συναντώνται είναι 1, εκτός από τα αντίστοιχα εξόνια για την περιοχή FIMAC της γραμμικής θέσης 1 στις πρωτεΐνες C6 και C7 (intron phase 2), που φαίνεται να είναι εξελικτικά μεταγενέστερες περιοχές, και δημιουργήθηκαν με διπλασιασμό εξονίου, σε μεταγενέστερα εξελικτικά στάδια. / The complement system is a key component of the innate immune system and links the innate and adaptive immunity. It consists of more than 35 soluble and membrane proteins that initially are found as inactivated proenzymes and they can be activated by a proteolytic cascade. All three pathways that activate the complement leads to the formation of a Membrane Attack Complex (MAC) that lyses the pathogenic microorganisms. Proteins that participate in the formation of MAC, belongs to the MACPF (MAC – Perforin) family. The FIMAC (Factor I Membrane Attack Complex) module plays significant role in the function of MACPF proteins. The Complement proteins 6 (C6) and 7 (C7) that are components of the MAC, include the FIMAC module in their sequences and it seems that this module is their binding region with C345C of C5b. Moreover, the FIMAC module exists in Complement Factor I (CFI), which is a serine protease (SP) and degrades C4b and C3b molecules. The carboxyl - terminal of FIMAC module binds allosteric with the SP region in CFI and seems to be important for the function of CFI as a serine protease. The model for the analysis of FIMAC module was based in Follistatin Domain (FD) of follistatin (FS) protein, which has been analyzed by crystallography. FIMAC and FD modules seem to have homologous sequences. The FD structure is a hybrid of an amino – terminal EGF (Epidermal Growth Factor) subdomain and of a carboxyl – terminal similar to ovomucoid subdomain, which is called KAZAL and is present in many serine protease inhibitors. The FD module is also present in the Agrin (AGRN) protein. AGRN is an extracellular matrix molecule released by the nerve and is critical for the formation of the neuromuscular junction. The subject of this work was the study of FIMAC and KAZAL modules concerning their function, structure and evolution. There were used data from biological databases and bioinformatic tools for analysis. The nucleotide and amino acid sequences of C6, C7, CFI, AGRN and FS genes from the organisms that were found (vertebrates and invertebrates), and more specific, FIMAC and KAZAL sequences, were the primary material of this study. There are differences in the length of the primary structures of FIMAC ( ̴ 78aa) and KAZAL ( ̴ 55aa) modules, except for some KAZAL modules of AGRN and FS proteins (>80aa). The secondary structures of FIMAC and KAZAL modules seem to be similar as both of them contain α-helix and β-sheet conformations. Simulation models of tertiary structure of both FIMAC and KAZAL modules revealed a common conformation of α-helix and β-sheet in space. The presence of cysteine residues are very conserved and seem to be important in FIMAC (8 – 10 Cys) and KAZAL (4 – 6 Cys) modules, although the concentration of cysteine residues in FIMAC modules are denser in amino – terminal region compared with their corresponding concentration in KAZALs, where they follow an equable distribution. Evolutionary, the genes that participate in all three pathways of complement activation and result in MAC formation, first appeared on chondrichthyes. Moreover, FIMAC and KAZAL modules included in CFI sequence found firstly on agnatha, on chondrichthyes in C6 sequences and on osteichthyes in C7 sequences. The presence of AGRN and FS genes were certified earlier in evolution on cephalochordates and platyelminthes of protostomes. As a result, it seems that the KAZAL modules of AGRN and FS proteins in invertebrates are the ancestors of all FIMAC and KAZAL modules. Nevertheless, new KAZAL modules appeared later during evolution of species. At the genomic level, exons corresponding to the FIMAC and KAZAL modules are highly conserved in different taxa. Intron phases of all exons corresponding to the FIMAC and KAZAL modules in all genes are 1, except for exons of FIMAC modules in first position of C6 and C7 genes (phase 2) that seem to be evolutionary posterior and were emerged by exon duplication, later in evolution.

No description available.

Epidermal homeostasis, including proper turnover of keratinocytes, plays important role in the barrier function and serine proteases and their inhibitors are the key players. Activated proteases cleave desmosomes in uppermost layer and thus shed the cells from the epidermal surface. Therefore the serine protease inhibitors are secreted in lower epidermal layers to prevent premature activation of proteases and consequent disruption of epidermal barrier. The most studied inhibitors in epidermis belong to Serine proteases inhibitors Kazal-type family (SPINK). This diploma thesis is aimed to investigate function of murine SPINK6 in epidermal compartment in vivo. To achieve this, the transgenic mice overexpressing mSPINK6 under modified human involucrin promoter was generated. Two of five transgenic lines exhibited higher expression of mSPINK6 at mRNA and protein levels. The mSPINK6 transgenic mice are viable with no apparent phenotype. The small but in most cases not significant differences were observed on microscopic level among mSPINK6 transgenic and wild type animals In conclusion, this work showed that mSPINK6 does not play major role in skin homeostasis but gains significant importance under specific challenges of epidermal barrier. Therefore mSPINK6 transgenic mice, in combination with other deletion or...