Au cours de ce travail de thèse, les deux approches permettant la caractérisation de nouveaux agents antileishmaniens ont été suivies : d’une part identification de molécules de synthèse originales et actives, par des méthodes de criblage, et d’autre part recherche de nouvelles cibles thérapeutiques leishmaniennes, faisant appel à des outils de biologie moléculaire, et de protéomique.Dans une première partie consacrée à l’optimisation du criblage antileishmanien de molécules de synthèse, nous avons notamment mis au point et validé une nouvelle méthode applicable aux formes promastigotes. Cette méthode reposant sur le principe de la bioluminescence, s’est avérée précise, rapide, répétable, sensible, automatisable et applicable à des isolats cliniques. Nous nous sommes également intéressés à la recherche d’une nouvelle méthode de criblage sur les formes amastigotes intracellulaires remplissant ces mêmes critères.Dans une seconde partie consacrée au lien entre la mort cellulaire programmée et les cystéines peptidases (métacaspase et Raptor) chez Leishmania, nous avons mis en évidence un lien possible entre métacaspase et autophagie chez L. infantum. De plus, nous avons identifié, in vivo, plusieurs substrats protéiques potentiels de ces peptidases : HSP70, ARN-Hélicase ATP-Dépendante et Lmjf09.1010 pour la métacaspase ; NDPKb et la protéine associée à l’ADN du kinétoplaste pour Raptor. Ces différents résultats ont permis de proposer un modèle conciliant les rôles possibles des cystéine-Peptidases métacaspase et Raptor au cours de l’autophagie et de l’apoptose chez Leishmania, rôles potentiellement dus au clivage de substrats protéiques spécifiques. / During this thesis work, two approaches affording the characterization of new antileishmanial agents were followed: on the one hand, the identification of new original antileishmanial synthetic drugs, through screening assays and on the other hand, research of new parasitic therapeutic targets by using molecular biology and proteomic tools. In the first part, dedicated to the optimization of the antileishmanial screening of synthetic compounds, we set up and validated a new bioluminescence-Based screening method for studying anti-Promastigote compounds. This method appears accurate, rapid, repeatable, sensitive and is also transposable to automats and usable on clinical isolates. In parallel, we investigated new screening protocols aiming at improving the screening in intracellular amastigotes which could meet the same criteria. In the second part focusing on the link between programmed cell death and cystein peptidases (metacaspase and Raptor) in Leishmania, our study first highlighted a possible link between metacaspase and autophagy in L. infantum. Moreover, we identified in vivo several potential subtrates for both peptidases: HSP70, ATP-Dependent RNA-Helicase and Lmjf09.1010 for the metacaspase; NDPKb and kinetoplast-DNA-Associated-Protein for Raptor. These results allowed us to propose a model reconciling the possible roles of cystein peptidases metacaspase and Raptor during autophagy and apoptosis in Leishmania, roles potentially due to the cleavage of specific proteic subtrates.

Na cascavel Crotalus durissus terrificus ocorre o armazenamento de longo prazo do esperma (LTSS), no trato genital da fêmea, durante o intervalo entre a cópula (outono) e a ovulação (primavera). Peptídeos e peptidases estão entre os principais componentes que influenciam a atividade espermática em mamíferos. O presente estudo objetivou caracterizar a presença de peptidases em C. d. terrificus, com esta função reconhecida em mamíferos e/ou que clivam peptídeos atuantes nesta função, bem como avaliar a hipótese do envolvimento destas peptidases na preservação dos espermatozóides desta serpente. O aspecto morfo-funcional dos espermatozóides foi comparado na presença dos peptídeos angiotensina II (AngII), arginina vasotocina (AVT), bradicinina (BK), peptídeo promotor da fecundação (FPP) e hormônio liberador de tireotrofina (TRH). Caracterizamos os efeitos de agentes quelante, tiol-redutor, cofator e inibidores, bem como dos peptídeos supracitados, sobre as atividades enzimáticas relacionadas, quais sejam: aminopeptidases ácida (APA), básica (APB), alanil sensível (APN-PS) e insensível à puromicina (APN-PI), cistil (CAP), dipeptidil-peptidase IV (DPPIV), piroglutamil tipo 1 (PAP-I) e prolil-imino- (PIP), bem como da prolil endopeptidase (POP), em frações solúvel (FS) e/ou de membrana solubilizada (FM) do sêmen proveniente do ducto deferente, assim como deste próprio tecido e dos tecidos uterino e vaginal de C. d. terrificus, ou seja, tecidos por onde passam ou armazenam-se os espermatozóides. Verificamos a variação sazonal destas mesmas atividades, nestes tecidos, incluindo o sêmen armazenado no útero durante o LTSS. O sêmen coletado do ducto deferente foi fracionado para avaliação da distribuição destas atividades peptidásicas. Nossos dados mostraram características de liquefação do sêmen e movimento dos espermatozóides da cascavel que os diferenciam do padrão humano. FPP com cálcio e BK melhoraram a preservação da viabilidade espermática, similarmente ao que ocorre em mamíferos. Em todos os tecidos e no sêmen as atividades APB, PIP e POP foram detectadas apenas da FS, enquanto as demais estão presentes em FS e FM, seguindo um padrão de distribuição observado na maioria dos tecidos de mamíferos. Amastatina e bestatina inibiram APB e APN, enquanto a diprotina A foi o inibidor mais eficiente para a DPPIV em FM. PAP-I e PIP foram inibidas, respectivamente, por bestatina e puromicina. Este perfil de inibição também é similar ao encontrado para as aminopeptidases em tecidos de mamíferos. Todas as atividades peptidásicas foram influenciadas por algum dos peptídeos estudados, sugerindo que tais peptídeos são substratos potenciais e/ou moduladores destas atividades na cascavel. As atividades APB e APN foram caracterizadas como metalopeptidases. APB, CAP e DPPIV foram inibidas por MnCl2. CAP e PAP-I foram caracterizadas como enzimas sulfidril-dependentes. As atividades APB, APN-PI e APN-PS predominaram, em relação às demais peptidases, em todas as estações e na maioria dos tecidos e no sêmen, sugerindo sua maior relevância na fisiologia reprodutiva da C. d. terrificus. Os níveis de todas as atividades peptidásicas estudadas variaram sazonalmente, sugerindo que sua ação moduladora sobre peptídeos susceptíveis está integrada ao ciclo reprodutivo desta serpente. O fracionamento do sêmen do ducto deferente revelou a presença de fluido seminal e espermatozóides, bem como de uma estrutura prostassoma-símile, até então somente identificada em mamíferos. Em todas estas frações há atividade peptidásica, predominando a APN-PI no prostassoma-símile e no fluido seminal, e APN-PS e APN-PI na FS e FM dos espermatozóides, caracterizando um envolvimento com a redução da motilidade espermática, tal qual ocorre em mamíferos. Concluimos que as atividades peptidásicas estudadas apresentam características sazonais e tecido-específicas que sugerem uma atuação relevante na preservação dos espermatozóides de C.d. terrificus. / In the rattlesnake Crotalus durissus terrificus occurs the long-term sperm storage (LTSS), in the female tract, during the interval between mating (autumn) and ovulation (spring). Peptides and peptidases are among the main components that influence the spermatic activity in mammals. The present study aimed to characterize the presence of peptidases in C. d. terrificus, that are well-recognized to exert this function and/or that have the ability to hydrolyze peptides that exert this function in mammals, as well to evaluate whether these peptidases are related to the preservation of spermatozoids in this snake. The morphological and functional characteristics of spermatozoids were compared in the presence of angiotensin II (AngII), arginine-vasotocin (AVT), bradykinin (BK), fertilization promoting peptide (FPP) and thyrotropin-releasing hormone (TRH). We have checked the effect of chelating and thiol-reducing agents, cofactor and inhibitors, as well the effect of aforementionated peptides on related enzyme activities such as acid (APA), basic (APB), puromycin-sensitive (APN-PS) and puromycin-insensitive alanyl (APN-PI), cystyl (CAP), pyroglutamyl type 1 (PAP-I) and prolyl-imino (PIP) aminopeptidases, and dipeptidyl-peptidase IV (DPPIV), as well as on prolyl endopeptidase (POP), in soluble (FS) and/or solubilized membrane-bound (FM) fractions of semen from vas deferens, of this own tissue, and vagina and uterus tissues of C. d. terrificus, i.e. tissues where spermatozoids pass through or where they are stored. The seasonal variation of these peptidase activities, in all tissues, including the semen stored in uterus during the LTSS, were evaluated. The semen from vas deferens was fractioned in order to know the distribution of these peptidase activities. The features of seminal liquefaction and movement of spermatozoids were different between the rattlesnake and human. Similar to mammals, FPP plus calcium and BK improved the preservation of the viability of spermatozoids from C. d. terrificus. In all tissues and semen, the APB, PIP and POP activities were detected only in FS, while others peptidases were present in FS and FM, following a similar pattern of distribution usually observed in mammalian tissues. Amastatin and bestatin inhibited APB and APN activities, while diprotin A was the most efficient inhibitor of DPPIV in FM. PAP-I and PIP activities were inhibited by bestatin and puromycin, respectively. This inhibition profile was similar to that of mammalian tissues. All peptidase activities were influenced at least by one of the peptides under study, suggesting these peptides as potential substrates and/or modulators for these peptidases of the rattlesnake. The APB and APN activities were characterized as metallopeptidases. APB, CAP and DPPIV were inhibited by MnCl2. CAP and PAP-I were characterized as sulfhydryl-dependent enzymes. The APB, APN-PI and APN-PS activities were predominant, in relation to the other examined peptidases, in all seasons and in most tissues and semen, suggesting their great relevance in the reproductive physiology of the C. d. terrificus. The levels of all studied peptidase activities were seasonally variable, suggesting that their modulator actions on susceptible peptides are integrated to the reproductive cycle of this snake. The fractionation of C. d. terrificus semen revealed the presence of seminal fluid and spermatozoids, as well a prostasome-like structure, until then identified only in mammals. In all of these fractions, there are peptidase activities, predominating the APN-PI in prostasome and seminal fluid, and the APN-PS and APN-PI in FS and FM of spermatozoids, suggesting their involvement in the reduction of the spermatic mobility, such as in mammals. In conclusion, the studied peptidase activities present seasonal and tissue-specific characteristics, which suggest a relevant role in the preservation of the spermatozoids of C. d. terrificus.

Protein synthesis is an essential process for all living organisms and is an effective major target for current antibiotics. Elongation factor Tu (EF-Tu) is a highly conserved and essential protein that functions during protein synthesis. EF-Ts interacts with EF-Tu to help maintain a functionally active state of EF-Tu required for cell growth. Although EF-Ts is essential for Escherichia coli, its sequence is poorly conserved. LepA is a highly conserved protein within bacteria and has a similar structure to EF-Tu. In spite of this, LepA has been shown to be non-essential under ideal conditions and the function of LepA still remains elusive. An analysis on the structurally unique aspects of LepA, EF-Tu and EF-Ts was performed here in an effort to gain an understanding on the functions of these proteins. This knowledge, in combination with their unique structural components will provide important tools in developing new and effective antibiotics. / xiii, 177 leaves : col. ill. ; 29 cm

Naegleria fowleri, a free-living ameba, is the causative agent of Primary Amebic Meningoencephalitis. Highly-pathogenic mouse-passaged amebae (Mp) and weakly-pathogenic axenically-grown (Ax) N. fowleri were examined for peptidase activity. Zymography and azocasein peptidase activity assays demonstrated that Mp and Ax N. fowleri exhibited a similar peptidase pattern. Prominent for whole cell lysates, membranes and conditioned medium from Mp and Ax amebae were the presence of an activity band of approximately 58kDa and 100 kDa bands susceptible to the action of cysteine and metallopeptidase inhibitors, respectively. Further roles of the peptidases during the invasion process were examined by in vitro invasion assays in the presence of inhibitors and Cysteine and metallopeptidase inhibitors were found to greatly reduce invasion through the ECM. This study establishes a functional linkage of the expressed peptidases to the invasion process, and these peptidases may serve as a candidate target for therapeutic management of N. fowleri infection.

La détermination du rôle des protéines est à l'origine de la plupart des études biologiques. Elle est encore plus d'actualité depuis l'avènement du séquençage des génomes qui fournit des protéines de rôle inconnu en grande quantité. Le séquençage de nouveaux génomes a permis, par ailleurs, l'obtention de données post-génomiques à haut débit comme les données transciptomique. Il a permis également la construction de données comme les profils phylogénétiques. L'approche que nous avons utilisée, mêlant statistiques et biologie, intègre ces données post-génomiques pour prédire puis valider le rôle de protéines protéolytiques chez Lactococcus lactis. Nous avons procédé en trois étapes réalisant, dans un premier temps, une inférence statistique des liens prédits entre protéines du lactocoque basée sur cinq sources de données distinctes : le graphe des voies métaboliques, des données transcriptomiques, des profils phylogénétiques, des distances entre gènes en paires de bases et des données protéomiques issues de gels 2D. Ces liens nous ont permis de émettre des hypothèses biologiques sur le rôle de protéines cibles. Dans un deuxième temps, nous avons réalisé des expériences de laboratoire pour valider les prédictions comparant la souche sauvage aux mutants de délétion de PepF et YvjB. La troisième étape de ce travail a consisté à réaliser une analyse de sensibilité dans le but de souligner les données qui ont contribuées le plus aux prédictions et de trouver un sous-ensemble de données aboutissant aux mêmes prédictions. Appliquée à deux enzymes protéolytiques potentielles du lactocoque, PepF et YvjB, cette approche nous a permis de vérifier leur participation dans l'export de protéines telle qu'elle avait été prédite par l'analyse statistique. Nous avons montré que PepF participe à l'export de protéines libérées dans le milieu extérieur (protéines sécrétées) en hydrolysant le peptide signal libéré par la signal peptidase I. PepF participe par ailleurs aussi à la synthèse de la paroi cellulaire et au métabolisme du pyruvate, fonctions aussi prédites par l'analyse statistique. YvjB participe à la mise en place et la maturation d'un autre groupe de protéines exportées, les lipoprotéines. Elle est nécessaire pour la localisation correcte de certaines lipoprotéines et participe au clivage de leur peptide signal. Notre approche s'est avérée très utile dans la mesure où elle permet d'émettre des hypothèses biologiques qui guident les validations expérimentales. Par ailleurs, cette démarche est applicable à tout organisme entièrement séquencé pour lequel suffisamment de données post-génomiques sont disponibles.

La ghréline est un peptide de 28 acides aminés, produit principalement par l’estomac et caractérisé par la présence d’un groupement octanoyl sur la sérine en position 3 (Kojima et al. 1999). La ghréline stimule la libération de l’hormone de croissance (GH) et régule la prise alimentaire et le métabolisme énergétique (Gualillo et al. 2003). Ces activités biologiques sont principalement médiées par le « growth hormone secretagogue receptor » (GHS-R). Deux sous-types de GHS-R, produits par épissage alternatif d’un même gène, ont été clonés : le GHS-R 1a, dont l’activation entraîne une libération de calcium via la formation d’inositol 1,4,5-trisphosphate (IP3), et le GHS-R 1b, qui ne semble pas lié à une activité biologique (Howard et al. 1996). La première partie de mon travail de thèse consistait en l’étude de la dégradation de la ghréline. La ghréline circule dans le sang principalement sous forme de des-acyl ghréline, une forme de ghréline dépourvue du groupement octanoyl qui ne se lie pas au GHS-R 1a. Peu d’études ont été réalisées sur le catabolisme de la ghréline. Les enzymes impliquées dans la dégradation de la ghréline étant des régulateurs importants de son activité biologique, le but de cette étude était d’identifier les sites de clivage et les enzymes impliquées dans la dégradation de la ghréline par du sérum, des sous-fractions plasmatiques et des homogénats de tissus. Nous avons montré qu’au contact de sérum humain et de rat, la ghréline est désoctanoylée, sans protéolyse. Dans le sérum humain, nous avons montré que la butyrylcholinestérase et la « platelet-activating factor acetylhydrolase » (PAF-AH), une phospholipase associée aux lipoprotéines de basse densité (LDL), sont impliquées dans ce phénomène (articles n°1 et n°2). En parallèle, nous avons montré que la ghréline peut être transportée dans la circulation sanguine par les lipoprotéines riches en triglycérides (TRL), les LDL, et les lipoprotéines de haute et de très haute densité (HDL et VHDL) (article n°2). Dans le sérum de rat, la désoctanoylation de la ghréline implique une carboxylestérase (article n°1). Au contact d’homogénats de tissus, la ghréline est dégradée à la fois par désoctanoylation et protéolyse N-terminale, suggérant la participation d’estérases et d’aminopeptidases. Nous avons identifié cinq sites de clivage dans la ghréline : entre les résidus Ser2-(acyl)Ser3 (dans l’estomac et le foie), (acyl ?)Ser3-Phe4 (dans l’estomac, le foie et le rein), Phe4-Leu5 (dans l’estomac et le rein), Leu5-Ser6 et Pro7-Glu8 (dans le rein) (article n°1). La deuxième partie de mon travail de thèse consistait à étudier l’expression et la production de ghréline par différentes lignées leucémiques (HEL, HL-60, THP-1, SupT1), par des leucocytes poly- et mononucléés et par des plaquettes sanguines, et à étudier l’effet de la ghréline sur la prolifération cellulaire. Pour cela, nous avons mis au point des dosages radioimmunologiques (RIA) permettant de quantifier et de distinguer les formes octanoylées et non octanoylées de la ghréline, et nous avons caractérisé en détail les anticorps SB801 et SB969 obtenus. Par HPLC en phase inverse suivie des RIAs, nous avons mis en évidence la présence de ghrélines octanoylée et non octanoylée dans chaque population de cellules. Plus de 80 % de la ghréline produite est octanoylée dans les cellules HEL, les leucocytes et les plaquettes. Nous avons montré que la ghréline endogène stimule la prolifération des cellules HEL de façon autocrine impliquant un récepteur encore non identifié, distinct du GHS-R 1a (article n°3). La ghréline et la des-acyl ghréline inhibent la prolifération des leucocytes mononucléés mais sont dépourvues d’effet sur les cellules HL-60, THP-1 et SupT1. Malgré la présence du GHS-R 1a dans les leucocytes mononucléés, cet effet pourrait être médié par un récepteur différent puisque la des-acyl ghréline exerce le même effet que la ghréline sur la prolifération (article n°4).

Thèse de doctorat : Sciences agronomiques : Vandoeuvre-les-Nancy, INPL : 2006. / Titre provenant de l'écran-titre. Bibliogr.

Thèse de doctorat : Procédés biotechnologiques et alimentaires : INPL : 2006. / Titre provenant de l'écran-titre. Bibliogr.

The translational GTPases elongation factor Tu (EF-Tu) and LepA modulate the dynamics of tRNA on the ribosome. EF-Tu facilitates the delivery of aminoacyl-tRNA (aa-tRNA) to the translating ribosome and LepA catalyzes the retro-translocation of tRNA•mRNA from the E- and P-sites of the ribosome back to the P- and A-sites. Although an increasing body of structural and biochemical information is available, little is known about the functional cycle of LepA during retro-translocation, the kinetics of EF-Tu dissociation from the ribosome and the rate of EF-Tu conformational change during aa-tRNA delivery. This thesis reports the successful construction and biochemical characterisation of a mutant form of EF-Tu from Escherichia coli ideal for the specific incorporation of fluorescent labels, enabling measurements pivotal for uncovering the rate of EF-Tu conformational change and dissociation from the ribosome. Furthermore, to determine structural components critical for LepA’s function, mutant versions of the protein were constructed and biochemically characterised. / xii, 127 leaves : ill. (some col.) ; 29 cm

LIMA, Magda Laiara Bezerra de. Análise ultraestrutural e molecular do desenvolvimento do integumento interno de sementes de Jatropha curcas L. 2017. 102 f. Tese (Doutorado em Bioquímica)-Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by Anderson Silva Pereira (anderson.pereiraaa@gmail.com) on 2017-05-02T19:58:45Z No. of bitstreams: 1 2017_tese_mlblima.pdf: 7882259 bytes, checksum: 57d42e334a50ab63d28f9035b1d8229b (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2017-05-17T18:58:34Z (GMT) No. of bitstreams: 1 2017_tese_mlblima.pdf: 7882259 bytes, checksum: 57d42e334a50ab63d28f9035b1d8229b (MD5) / Made available in DSpace on 2017-05-17T18:58:34Z (GMT). No. of bitstreams: 1 2017_tese_mlblima.pdf: 7882259 bytes, checksum: 57d42e334a50ab63d28f9035b1d8229b (MD5) Previous issue date: 2017 / Jatropha curcas L. is a species of oleaginous plant that arouses economic interest because it is an alternative source for biodiesel production although little is known about the biology of the development of this plant organism. This knowledge can serve as a basis for the domestication of this species and the discovery of probable solutions for seed toxicity due to the presence of the phorbol esters in the seeds, for example. J. curcas seeds are made up by the embryo, endosperm as well as the surrounding seed coat. The latter is derived from the development of both outer (oi) and inner (ii) integuments. It is known that the ii may act as a transient storage tissue providing carbon and nitrogen sources for the development of the filial tissues, endosperm and embryo. Scientific reports indicate that during ii development the cells undergo programmed cell death (PCD). The aimed at observing the changes in the ultrastructure of the ii during its development and confirm the PCD characteristics by assessing the gene expression pattern for seven different hydrolases (arabinofuranosidase, glucanase, peptidase cysteine tailed RDEL, peptidase cysteine tailed KDEL, aspartic peptidase, serine peptidase and subtilisin) and two inhibitors (inhibitor peptidase cysteine and serpin). The stages used in this study (ovule and seeds at 5, 10 and 25 DAP) were selected based on the morphological changes in the ii observed in light microscopy. The vacuolization and cell expansion were the most conspicuous characteristics observed in the early development of the ii. Furthermore, the first related morphological changes related to PCD were observed at 5 DAP, including rising the periplasmic spaces and ricinossomos and collapse of tonoplasto. In later stages, at 10 and 25 DAP, it was observed enlargement of the cell volume, followed by less dense cytoplasm, presence of peroxisomes, lobed nucleus, condensed chromatin, invaginations and disruption of the plasma membrane and finally cell wall breakdown as well as the rising of gerontoplasts (25 DAP). In the early stages (ovule, 5 and 10 DAP) the expression pattern of genes encoding cysteine peptidase tailed KDEL, serine peptidase and subtilisin it was higher at 10 DAP if compared to the calibrator (ovule). In the ii proximal region of seeds with 25 DAP the expression of all peptidases (other than aspartic peptidase), the arabinofuranosidase and glucanase was statistically superior when compared to the distal region. The correlation of the ultrastructural findings and the transcriptional expression pattern indicates that the occurrence of CCM in the internal integument can act in the supply of carbon and nitrogen sources for the development of the embryo and endosperm. / Jatropha curcas L. é uma espécie de planta oleaginosa que desperta interesse econômico por ser uma fonte alternativa para a produção de biodiesel muito embora, pouco se sabe sobre a biologia do desenvolvimento desse organismo vegetal. Esses conhecimentos podem servir de base para a domesticação desta espécie e a descoberta de prováveis soluções para toxicidade das sementes devido a presença dos ésteres de forbol nas sementes, por exemplo. As sementes de J. curcas são constituídas pelo embrião e endosperma, ambos envolvidos pela testa e o tegma. Estes últimos são provenientes do desenvolvimento dos integumentos externo (ie) e interno (ii). É sabido que o ii pode atuar como tecido de armazenamento transeunte de fontes de carbono e nitrogênio para o desenvolvimento dos tecidos filiais, endosperma e embrião. Relatos científicos indicam que durante o desenvolvimento do ii as células sofrem morte celular programada (MCP). Buscou-se observar as modificações na ultraestrutura do ii em decorrência do seu desenvolvimento; além de validar as características da MCP por meio da avaliação do padrão de expressão de sete hidrolases (arabinofuranosidase, glucanase, peptidase cisteínica com cauda RDEL, peptidase cisteínica com cauda KDEL, peptidase aspártica, peptidase serínica e subtilisina) e dois inibidores de peptidase (inibidor de peptidase cisteínica e serpina). Além do óvulo, os estágios utilizados neste estudo (sementes aos 5, 10 e 25 DAP) foram selecionados com base nas modificações morfológicas do ii obtidas através da análise histológica. A vacuolização e a expansão celular foram as características mais conspícuas observadas no início do desenvolvimento do ii. Além disso, as primeiras mudanças morfológicas relacionadas a MCP foram observadas aos 5 DAP, incluindo, surgimento dos espaços periplasmáticos, aparecimento dos ricinossomos, e colapso do tonoplasto. A sequência do processo degradativo foi observada nos estágios mais avançados (10 e 25 DAP) com expansão do volume celular, acompanhado de citoplasma menos denso, presença de peroxissomos, núcleo lobado, cromatina condensada, invaginações e ruptura da membrana plasmática e, finalmente a ruptura da parede celular, além do surgimento dos gerontoplastos (25 DAP). Nos estágios iniciais (óvulo, 5 e 10 DAP) o padrão de expressão relativa dos genes que codificam para peptidase cisteínica KDEL, peptidase serínica e subtilisina foi maior aos 10 DAP quando comparados com o óvulo. Na região proximal do ii das sementes com 25 DAP a expressão de todas as peptidases (exceto peptidase aspártica), a arabinofuranosidase e glucanase foi estatisticamente superior quando comparada à região distal desse mesmo estágio. A correlação dos achados ultraestruturais e o padrão de expressão transcricional indica que a ocorrência da MCP no integumento interno pode atuar no fornecimento de fontes de carbono e nitrogênio para o desenvolvimento do embrião e endosperma.