Auswahl der wissenschaftlichen Literatur zum Thema „Famille M1“

Abstract Cross-reactive memory T cells specific to previously encountered influenza A contribute to the lymphoproliferation characteristics of EBV-associated infectious mononucleosis (IM). We showed that cross-reactive CD8 T cell responses commonly occur during EBV infection. Strong cross-reactive T cell responses specific to influenza M1 (Flu-M1) and EBV-specific lytic epitope EBV-BRLF-1 have been observed in 10/10 HLA-A2+ IM patients. We also detected cross-reactive T cell responses that recognized both Flu-M1 and lytic EBV-BMLF-1 epitopes in 14/26 HLA-A2+ IM patients. In addition, 8/19 IM patients demonstrated some intra-viral cross-reactivity between EBV BRLF-1 and BMLF-1 specific epitopes. Cross-reactive T cell responses between Flu-M1 and EBV-BRLF-1/EBV-BMLF-1 epitopes were observed in all IM patients but at different time points after infection. Our analyses showed that Flu-M1 and EBV-BMLF-1 used few Vbeta TCR families that were stable overtime. However, EBV-BRLF-1-specific Vbeta TCR repertoire used multiple Vbeta families that varied between individuals and varied during the time course of IM in the same patient. Disease severity of IM directly correlated with percentage of Flu-M1-specific CD8 T cells but did not correlate with EBV-BMLF-1 or BRLF-1-specific responses.

This study aims to expand our understanding of the genetic architecture of crown morphology in the human diphyodont dentition. Here, we present bivariate genetic correlation estimates for deciduous and permanent molar traits and evaluate the patterns of pleiotropy within (e.g., m1–m2) and between (e.g., m2–M1) dentitions. Morphology was observed and scored from dental models representing participants of an Australian twin and family study (deciduous n = 290, permanent n = 339). Data collection followed Arizona State University Dental Anthropology System standards. Genetic correlation estimates were generated using maximum likelihood variance components analysis in SOLAR v.8.1.1. Approximately 23% of deciduous variance components models and 30% of permanent variance components models yielded significant genetic correlation estimates. By comparison, over half (56%) of deciduous–permanent homologues (e.g., m2 hypocone–M1 hypocone) were significantly genetically correlated. It is generally assumed that the deciduous and permanent molars represent members of a meristic molar field emerging from the primary dental lamina. However, stronger genetic integration among m2–M1/M2 homologues than among paired deciduous traits suggests the m2 represents the anterior-most member of a “true” molar field. The results indicate genetic factors act at distinct points throughout development to generate homologous molar form, starting with the m2, which is later replaced by a permanent premolariform crown.

AbstractErdős and Szekeres [‘Some number theoretic problems on binomial coefficients’, Aust. Math. Soc. Gaz.5 (1978), 97–99] showed that for any four positive integers satisfying m1+m2=n1+n2, the two binomial coefficients (m1+m2)!/m1!m2! and (n1+n2)!/n1!n2! have a common divisor greater than 1. The analogous statement for k-element families of k-nomial coefficients (k>1) was conjectured in 1997 by David Wasserman.Erdős and Szekeres remark that if m1,m2,n1,n2 as above are all greater than 1, there is probably a lower bound on the common divisor in question which goes to infinity as a function of m1 +m2 . Such a bound is obtained in Section 2.The remainder of this paper is devoted to proving results that narrow the class of possible counterexamples to Wasserman’s conjecture.

The synthesis of racemic substituted 7-amino-5,7,8,9-tetrahydrobenzocyclohepten-6-one hydrochlorides was optimized to enhance reproducibility and increase the overall yield. In order to investigate their specificity, series of enzyme inhibition assays were carried out against a diversity of proteases, covering representative members of aspartic, cysteine, metallo and serine endopeptidases and including eight members of the monometallic M1 family of aminopeptidases as well as two members of the bimetallic M17 and M28 aminopeptidase families. This aminobenzosuberone scaffold indeed demonstrated selective inhibition of M1 aminopeptidases to the exclusion of other tested protease families; it was particularly potent against mammalian APN and its bacterial/parasitic orthologues EcPepN and PfAM1.

Proteolytic enzymes, also known as peptidases, are critical in all living organisms. Peptidases control the cleavage, activation, turnover, and synthesis of proteins and regulate many biochemical and physiological processes. They are also involved in several pathophysiological processes. Among peptidases, aminopeptidases catalyze the cleavage of the N-terminal amino acids of proteins or peptide substrates. They are distributed in many phyla and play critical roles in physiology and pathophysiology. Many of them are metallopeptidases belonging to the M1 and M17 families, among others. Some, such as M1 aminopeptidases N and A, thyrotropin-releasing hormone-degrading ectoenzyme, and M17 leucyl aminopeptidase, are targets for the development of therapeutic agents for human diseases, including cancer, hypertension, central nervous system disorders, inflammation, immune system disorders, skin pathologies, and infectious diseases, such as malaria. The relevance of aminopeptidases has driven the search and identification of potent and selective inhibitors as major tools to control proteolysis with an impact in biochemistry, biotechnology, and biomedicine. The present contribution focuses on marine invertebrate biodiversity as an important and promising source of inhibitors of metalloaminopeptidases from M1 and M17 families, with foreseen biomedical applications in human diseases. The results reviewed in the present contribution support and encourage further studies with inhibitors isolated from marine invertebrates in different biomedical models associated with the activity of these families of exopeptidases.

ABSTRACT Helicobacter pylori, a gram-negative bacterium associated with gastritis, peptic ulceration, and gastric adenocarcinoma in humans, secretes a protein toxin, VacA, that causes vacuolar degeneration of epithelial cells. Several different families of H. pylori vacA alleles can be distinguished based on sequence diversity in the “middle” region (i.e., m1 and m2) and in the 5′ end of the gene (i.e., s1 and s2). Type s2 VacA toxins contain a 12-amino-acid amino-terminal hydrophilic segment, which is absent from type s1 toxins. To examine the functional properties of VacA toxins containing this 12-amino-acid segment, we analyzed a wild-type s1/m1 VacA and a chimeric s2/m1 VacA protein. Purified s1/m1 VacA from H. pylori strain 60190 induced vacuolation in HeLa and Vero cells, whereas the chimeric s2/m1 toxin (in which the s1 sequence of VacA from strain 60190 was replaced with the s2 sequence from strain Tx30a) lacked detectable cytotoxic activity. Type s1/m1 VacA from strain 60190 formed membrane channels in a planar lipid bilayer assay at a significantly higher rate than did s2/m1 VacA. However, membrane channels formed by type s1 VacA and type s2 VacA proteins exhibited similar anion selectivities (permeability ratio, PCl/PNa = 5). When an equimolar mixture of the chimeric s2/m1 toxin and the wild-type s1/m1 toxin was added to HeLa cells, the chimeric toxin completely inhibited the activity of the s1/m1 toxin. Thus, the s2/m1 toxin exhibited a dominant-negative phenotype similar to that of a previously described mutant toxin, VacA-(Δ6–27). Immunoprecipitation experiments indicated that both s2/m1 VacA and VacA-(Δ6–27) could physically interact with a c-myc epitope-tagged s1/m1 VacA, which suggests that the dominant-negative phenotype results from the formation of heterooligomeric VacA complexes with defective functional activity. Despite detectable differences in the channel-forming activities and cytotoxic properties of type s1 and type s2 VacA proteins, the conservation of type s2 sequences in many H. pyloriisolates suggests that type s2 VacA proteins retain an important biological activity.

AbstractThe article colligates data of studies on the variability of quantitative and qualitative traits in mutant sunflower M1-M3 generations affected by dimethyl sulfate (DMS) (0.01, 0.05 %) and gamma rays (120; 150 Gy), frequencies and range of mutations in M2 and their inheritance in mutant families, chromosome aberrations in meiosis, as well as on the breeding and genetic value of induced mutants and possibilities of their use in breeding. The methodical peculiarities of the mutational breeding of the cross-pollinating crop were defined, and new mutants with changed features were created.Investigating new homozygous self-pollinated sunflower lines, we observed a more negative mutagenic impact of gamma irradiation (120 and 150 Gy) on the germinability of M1 sunflower seeds in the field compared with the DMS effect (0.01 and 0.05 %). The field germinability of DMS-treated seeds was 83–87 % vs. 11–15 % of gamma-irradiated ones.The mutagenic effect of gamma rays (120 and 150 Gy) on M1 meiosis was shown to be stronger than that of DMS (0.01 and 0.05 %). The percentage of cells with alterations varied within 15.79–18.78 % (120 Gy) and 20.38–25.26 % (150 Gy) compared to 0–0.16 % in the control.The effect of gamma rays on the frequency of morphoses in M1 was stronger, in particular, after exposure to 120 Gy or 150 Gy of gamma irradiation, the number of plants with alterations was 43 %, whereas after DMS treatment (0.01 and 0.05 %) this parameter averaged 27–28 %.We determined the inheritance of mutations of quantitative and qualitative traits, which are important for breeding, in mutant M2 families and selected mutant families with inherited altered traits that can be considered as mutations. Among the best mutations, there are morphological mutants with marker traits, mutants with increased content of oil in seeds, increased 1000-seed weight, increased contents of behenic, linoleic and palmitoleic acids as well as with absolute resistance to downy mildew.

AbstractMetallo-aminopeptidases (mAPs) control many physiological processes. They are classified in different families according to structural similarities. Neutral mAPs catalyze the cleavage of neutral amino acids from the N-terminus of proteins or peptide substrates; they need one or two metallic cofactors in their active site. Information about marine invertebrate’s neutral mAPs properties is scarce; available data are mainly derived from genomics and cDNA studies. The goal of this work was to characterize the biochemical properties of the neutral APs activities in eight Cuban marine invertebrate species from the Phyla Mollusca, Porifera, Echinodermata, and Cnidaria. Determination of substrate specificity, optimal pH and effects of inhibitors (1,10-phenanthroline, amastatin, and bestatin) and cobalt on activity led to the identification of distinct neutral AP-like activities, whose biochemical behaviors were similar to those of the M1 and M17 families of mAPs. Additionally, M18-like glutamyl AP activities were detected. Thus, marine invertebrates express biochemical activities likely belonging to various families of metallo-aminopeptidases.

The vacuolating cytotoxin of Helicobacter pylori (VacA) is known to cause cell damage to mammalian cells and is suspected to give rise to gastric epithelial lesions that might lead to peptic ulcer disease. As shown recently, the gene encoding VacA exhibits genetic variation, with three different families of signal sequences (s1a, s1b, and s2) and two families of midregion sequences (m1 and m2). In order to investigate the relationship between the presence of specificvacA genotypes and peptic ulceration, the vacAgenotypes of 158 clinical isolates of H. pylori were determined. The study group consisted of 106 patients with duodenal ulceration; 52 patients with nonulcer dyspepsia (NUD) were used as controls. H. pylori of genotype s1 was isolated from 96% of the patients with ulcerations, whereas genotype s2 was only present in 4%, indicating a strong correlation between thevacA genotype and peptic ulceration (P < 0.001). In contrast, 31% of the patients from the NUD control group were infected with strains of vacA genotype s2. Particular midregion genotypes (m1 and m2) were not associated with clinical manifestations. The midregions from 18% of the isolates could not be classified by the proposed scheme. DNA sequencing revealed high homology between the untypeable midregions and that of genotype m1, with multiple base pair exchanges, some affecting the primer annealing site. Compared to those of m1 and m2 alleles, the divergent midregions from untypeable strains showed clustering, indicating the presence of a further subfamily of sequences in the midregion of vacA in German isolates, for which we propose the term “m1a.” A new specific primer that we designed for typing m1a isolates might be useful in other studies.

ABSTRACT Several different families of vacuolating toxin (vacA) alleles are present in Helicobacter pylori, and they encode products with differing functional activities. H. pyloristrains containing certain types of vacA alleles have been associated with an increased risk for peptic ulcer disease. In this study, we tested serum samples and gastric juice from 19 H. pylori-negative and 39 H. pylori-positive patients for enzyme-linked immunosorbent assay reactivity with two different types of VacA antigens (types s1/m1 and s2/m2), which were purified from H. pylori 60190 and 86-338, respectively. Both antigens were recognized better by serum immunoglobulin G (IgG) fromH. pylori-positive persons than by serum IgG from H. pylori-negative persons (P < 0.01). The s1/m1 VacA antigen was better recognized by sera from patients carryingvacA type s1/m1 strains than by sera from patients carryingvacA type s2/m2 or s1/m2 strains (P < 0.01). Conversely, the s2/m2 VacA antigen was better recognized by sera from patients carrying type s2/m2 or s1/m2 strains (P= 0.03). Serum IgG anti-VacA antibodies were present more frequently in patients carrying type s1/m1 strains than in other H. pylori-positive patients (P = 0.0002). In addition, the highest levels of IgA anti-VacA antibodies were detected in the gastric juice of patients carrying type s1/m1 strains. These data indicate that different VacA isoforms have distinct antigenic properties and that multiple forms of VacA elicit antibody responses inH. pylori-positive humans.

Les aminopeptidases de la famille M1 sont des protéases qui catalysent l’hydrolyse d’une liaison peptidique en position N-terminale. Ce sont des métalloprotéases avec un ion zinc dans leur site actif conservé dans tous les membres de cette famille de protéine. Ces enzymes sont impliqués dans de nombreux processus physiologiques normaux, mais également dans des désordres métaboliques, tels que la progression tumorale, des maladies auto-immunes, ainsi que dans des infections virales, bactériennes et parasitaires. Pour ces raisons, ces aminopeptidases sont considérées comme des cibles thérapeutiques potentielles pour traiter ou diagnostiquer diverses maladies. En 2006, le laboratoire a découvert le châssis moléculaire de type 3-amino-2-benzosubérone inhibant puissamment et sélectivement et un des membres de cette famille d’aminopeptidases, à savoir l’APN. La conception et la synthèse des dérivés de ce châssis moléculaire comme inhibiteurs potentiels et sélectifs pour cinq autres membres de la famille M1 (APN, ERAP1/2, IRAP et PfA-M1) est au cœur de ce travail. Des études pharmacologiques, pharmacocinétiques jusqu’aux essais précliniques ont été menées et leurs résultats seront présentés dans le cas de l’inhibition de PfA-M1
Aminopeptidases of the M1 family are proteases that catalyze the hydrolysis of a peptide bond in the N-terminal position. These are metalloproteases with a zinc ion in their active site conserved in all members of this protein family. These enzymes are involved in many normal physiological processes, but also in metabolic disorders, such as tumor progression, autoimmune diseases, as well as in viral, bacterial and parasitic infections. For these reasons, these aminopeptidases are considered potential therapeutic targets for treating or diagnosing various diseases. In 2006, the laboratory discovered the powerful and selectively inhibiting 3-amino-2-benzosuberone molecular chassis and one of the members of this family of aminopeptidases, namely the APN. The design and synthesis of derivatives of this molecular chassis as potential and selective inhibitors for five other members of the M1 family (APN, ERAP1 / 2, IRAP and PfA-M1) is at the heart of this work. Pharmacological, pharmacokinetic and preclinical studies have been conducted and their results will be presented in the case of PfA-M1 inhibition

L’aminopeptidase B (Ap-B ; EC 3. 4. 11. 6) hydrolyse les acides aminés basiques en N-terminal de peptides et est impliquée dans la maturation d’hormones et probablement de neuropeptides selon un mécanisme original récemment mis en évidence. À ce jour, un seul de ses substrats physiologiques a été identifié, le glucagon qui, de par l’action successive de la NRD convertase puis de l’Ap-B, est transformé en miniglucagon. Ces deux hormones participent à la régulation de l’homéostasie du glucose chez les Mammifères. Une autre particularité de l’Ap-B est de présenter, in vitro, une activité résiduelle de type e��poxyde hydrolase, capable d’hydrolyser le leucotriène A4 (LTA4) en leucotriène B4, un médiateur lipidique de l’inflammation. A l’inverse, son plus proche parent phylogénétique, la Leucotriène A4 Hydrolase (LTA4H ; EC 3. 3. 2. 6) possède, en plus de son activité époxyde hydrolase, une activité aminopeptidase toutefois moins spécifique. Une analyse structurale et fonctionnelle de l’Ap-B est nécessaire pour mieux comprendre les mécanismes catalytiques de l’enzyme et pour parvenir à la conception d’inhibiteurs spécifiques, dont l’utilisation devrait nous éclairer sur l’ensemble de ses fonctions in vivo. Nous avons mis au point un système d’expression et de purification de l’Ap-B chez E. Coli avant d’entreprendre une analyse fonctionnelle par mutagenèse dirigée d’un certain nombre de résidus potentiellement impliqués dans l’activité de l’enzyme. Ces acides aminés ont été identifiés par alignement des séquences des protéines de la famille M1 à laquelle appartiennent l’Ap-B et la LTA4H. Cette étude a, par ailleurs, permis d’identifier 3 sous-familles distinctes d’aminopeptidases. Cette famille M1 est caractérisée par la présence d’un motif de fixation du cation Zn2+ de type HEXXHX18E. Les mutations des résidus de ce motif conduisent à une perte totale de l’activité de l’Ap-B, confirmant ainsi le rôle essentiel de ces résidus dans le mécanisme catalytique. La famille M1 est également caractérisée par la présence d’un second motif consensus (GXMEN). Nous avons également muté l’ensemble des résidus de ce second motif. Si les mutations des acides aminés M, E et N conduisent à une perte de l’activité, les mutations du résidu G en sérine ou proline donnent naissance à deux protéines actives. Le mutant G298S a des propriétés proches de l’enzyme sauvage, tandis que le mutant G298P présente une modification de sa spécificité de substrat (clivage de R, K, P et A), de son profil d’inhibition et de son activité en présence d’ions Cl-. L’analyse de ces mutants par dichroïsme circulaire et spectrométrie de fluorescence montre que les structures globales de ces enzymes ne semblent pas perturbées. Dix autres acides aminés ont également été mutés et leur étude fonctionnelle est en cours. Les similitudes structurales et fonctionnelles de l’Ap-B et de la LTA4H dont la structure 3D est connue, nous a permis de construire un modèle moléculaire de la structure de l’Ap-B. Ce modèle a été utilisé pour, d’une part, essayer de comprendre le rôle du résidu Glycine du motif GXMEN et, d’autre part, pour mettre en évidence un certain nombre de différences entre l’Ap-B et la LTA4H, en particulier au niveau du site actif et des propriétés potentielles d’interactions protéine-protéines de l’Ap-B. En parallèle, nous avons mis au point un second système d’expression et de purification de l’Ap-B à partir d’un vecteur baculovirus et de cellules d’insectes. Ce système nous permet d’obtenir une quantité suffisante d’enzyme pour mettre au point les conditions de cristallogenèse de la protéine
Aminopeptidase B (Ap-B ; EC 3. 4. 11. 6) cleaves basic residues at the N-terminus of peptides and participates in hormone and neuropeptide processing through an original mechanism recently described. The only known physiological substrate of Ap-B is the glucagon, which is processed into miniglucagon by NRD convertase and Ap-B. In mammals, these hormones are involved in the regulation of glucose homeostasis. One second characteristic of Ap-B is that the enzyme exhibits, in vitro, a residual ability to hydrolyze leukotriene A4 (LTA4) into the pro-inflammatory lipid mediator leukotriene B4. Inversely, LTA4 hydrolase (LTA4H ; EC 3. 3. 2. 6), the closest homologous protein of Ap-B, possesses, besides an epoxyde hydrolase activity, an aminopeptidase activity of broader specificity. Both proteins belong to the M1 family of Zn2+-aminopeptidases. A structure-function analysis is needed for a detailed understanding of the enzymatic mechanisms of Ap-B and to aid in the design of inhibitors, which could be used to determine its whole in vivo functions. In order to carry out a functional analysis by site-directed mutagenesis, we developed an expression system and a purification procedure of Ap-B using E. Coli. Amino acid residues potentially involved in the aminopeptidase activity are identified using alignments of primary structures of proteins from the M1 family. This study allowed us to identify 3 distinct M1 sub-families. This M1 family is characterized by the presence of a HEXXHXn=18E Zn2+- binding motif. Mutations of these residues lead to a total loss of activity and confirm their essential roles in catalysis. The major part of the M1 family members (Ap-B, LTA4H,…) constitutes the main sub-family and possesses a second consensus motif (GXMEN). Mutations of the M, E or N residues also lead to a total loss of aminopeptidase activity. Surprisingly, replacement of the conserved glycine into a serine or a proline created two new active enzymes. The G298S mutant exhibits properties similar to the wild type enzyme, whereas the G298P mutant shows a modification of its substrate specificity (recognizing R, K, P and A), its inhibition profile and its behavior towards Cl-. Fluorescence spectroscopy and circular dichroïsm analyses did not show any fundamental modification in the mutant structures. Ten other residues were mutated in the Ap-B primary structure and their functional studies are in progress. The sequence and catalytic similarities shared between Ap-B and LTA4H and the determination of the X-ray structure of LTA4H led us to build a 3D structural model of Ap-B. This model was used, on one hand, to better understand the enzymatic role of the glycine residue of the GXMEN motif and, on the other hand, to highlight functional differences between Ap-B and LTA4H, particularly at the level of their active site and of their proteinprotein interaction properties. In parallel, we also developed a second system of expression and purification of Ap-B using baculovirus and insect cells. This system allows us to purify a sufficient amount of protein for crystallogenesis assays

Intracellular protein degradation is required for maintaining the cellular proteome and regulating cellular processes. This pathway involves proximal ATP-dependent proteases that unfold and translocate proteins targeted for degradation into catalytic chambers. The large peptides produced are further cleaved by ATP independent endopeptidases, aminopeptidases and carboxypeptidases to release free amino acids. Lon and Clp are the key ATP-dependent proteases in prokaryotes and 26S proteasomes in eukayotes. In general, enzymes involved in the distal processing of peptides are ATP-independent, display greater redundancy and their orthologs are present in most organisms. The aim of the present study was to generate biochemical and functional insights on the ATP-independent enzyme, Peptidase N (PepN), which belongs to the M1 family. Previous studies in our laboratory identified Escherichia Coli PepN, to harbor both amino and endopeptidase activitities. In addition, it is responsible for the cleavage of majority of aminopeptidase substrates in E. Coli and is known to be involved in Sodium salicylate(NaSal)-induced stress. The present study consists of four parts. First, intracellular proteolysis plays an important role for virulence in pathogens. Therefore, it becomes important to study the biochemical properties and roles of enzymes involved in protein degradation. In this direction, a study was initiated to characterize the biochemical properties of Peptidase N from Salmonella enterica serovar Typhimurium(S. typhimurium). To study the contribution of PepN to the overall cystosolic protein degradation in S.typhimurium, a targeted deletion in pepN was generated. Cystosolic lysates of S. typhimurium wild type(WT) and ΔpepN strains were examined for their ability to cleave a panel of aminopeptidase and endopeptidase substrates. The ΔpepN strain displayed greatly reduced cleavage of nine out of a total of thirteen exopeptidase substrates, demonstrating a significant contribution of PepN to cytosolic aminopeptidase activity. S. typhimurium PepN also cleaved the endopeptidase substrate Suc-LLVY-AMC, similar to E. Coli PepN. To understand the physiological role of PepN, WT and ΔpepN were subjected to different stress conditions. During nutritional downshift in combination with high temperature stress, the growth of ΔpepN was significantly reduced compared to WT. Importantly, the PepN overexpressing strains grew better than WT, demonstrating an enhanced ability to overcome this stress combination. The above study clearly underscores the importance of PepN, to play distinct roles during stress. The significance of this study lies in understanding the biochemical and functional properties of a M1 family member from a pathogenic organism. Second, peptidases belonging to the M1 family are widely distributed with orthologs found across different kingdoms. The key amino acids in the catalytic domain are conserved in this family. However, amino acids present in the C-termini are variable and the three available crystal structures of M1 family members display distint differences in organization of this domain. To investigate the functional role of C-termini, progressive deletions were generated in PepN from E.Coli and Tricorn interacting factor F2 from Thermoplasma acidophilum(F2). Catalytic activity was partially reduced inPepN lacking four aa from C-terminus (PepNΔC4) whereas it is greatly reduced in F2 lacking ten amino acids from C-terminus(F2ΔC10) or eleven amino acids from PepN (PepNΔC11). To understand the mechanistic reasons involved, biochemical and biophysical studies were performed on purified WT and C-termini deleted proteins. Increased binding to 8-amino- 1- naphthalene sulphonic acid (ANS) was observed for all C-termini deleted proteins revealing greater numbers of surface exposed hydrophobic amino acids. Further, trypsin sensitivity studies demonstrated that mutant proteins were more sensitive compared to WT. Notably, expression of PepNΔC4, but not PepNΔC11, in E ColiΔpepN increased its ability to resist nutritional and high temperature stress, demonstrating a physiological role for the C-terminus. Together, these studies reveal involvement of distal amino acids in the C-termini of two distant M1 family members in repressing the exposure of apolar residues and enhancing enzyme function. Third, the crystal structure of E. coliPepN displayed the presence of Zn2+. To study the role of metal cofactor, apo-PepN was isolated by chelating the holoenzyme with 1,10-phenanthroline. Among different metals tested, only Zn2+ rescued the greatly reduced catalytic activity of the apo-PepN. Further confirmatory studies were performed using pepN mutants in the conserved GXMEN and HEXXH motifs. No major structural differences were observed in purified mutants(E264A, H297A, and E298A) using circular dichroism (CD) and intrinsic fluorescence studies; however, they lacked catalytic activity. These studies clearly demonstrate that Zn2+ was essential for catalysis but not for the overall structural integrity of PepN. Estimation of the Zn2+ content by atomic absorption spectrometry demonstrated that the WT contained one molecule of zinc per molecule of enzyme. Similar results were obtained in purified proteins of E264A and E298A. residues involved in catalysis. However the Zn2+ amount was greatly reduced in H297A, which is involved in Zn2+ binding. Further, the in vivo role of metal cofactor and catalyis were studied during two established stress conditions. Over expression of the mutants, unlike WT, was unable to rescue the growth of ΔpepN during nutritional down shift and high temperature stress. These results demonstrate that E264, H297 and E298 were required for PepN function during nutritional downshift and high temperature stress. However during NaSal-induced stress condition, overexpression of WT or mutants reduced growth of ΔpepN, demonstrating that PepN function was independent of catalytic activity or metal cofactor. Further studies identified the YL motif, which is conserved in all members of the M1 family, to play a role during NaSal-induced stress. Over expression of Y185F or L186Q did not modulate catalytic activity although growth reduction of ΔpepN in the presence of NaSal was compromised. To understand the mechanisms by which the YL motif plays a role during this condition, Y185F and L186Q mutant proteins were purified. In vitro, both mutant proteins were found to aggregate at a lower temperature and their catalytic activities were more sensitive to temperature, compared to WT. Steady state analysis of WT, Y185F and L186Q were performed to study the modulation of PepN amount during stress conditions. Steady state amounts of Y185F and L186Q mutant proteins were greatly decreased compared to WT, during NaSal-induced stress. Most likely, the lowered amounts of Y185F and L186Q mutant proteins contribute to growth advantage during NaSal-induced stress. Thus, the YL motif in E. Coli PepN reduces protein aggregation and enhances the structural integrity of PepN during selective stress conditions in vivo. In summary, this study clearly identifies metal cofactor and peptidase-dependent and –independent motifs to play distinct functional roles in PepN. Fourth, the crystal structures of known M1 family members have shown that the catalytic domain and mechanism of action are similar. To identify novel residues that may modulate the catalytic activity of PepN, multiple sequence alignment of important M1 family members were performed. The alignment identified a subset of M1 family members, including PepN, containing an aspargine residue which is present two amino acids before glycine in the GAMEN motif. A closer investigation of thecrystal structure of PepN revealed an interaction between N259(Catalytic domain) with Q821 (C-terminal domain). To understand the functional role of this interaction, site-specific mutants were generated: N259D, Q821E and a double mutant, N259D & Q821E. Spectroscopic studies did not reveal any significant differences with respect to global structure or protein stability between purified WT and mutant enzymes. Also, binding to substrates by mutant enzymes was not affected as judged by Km values. However, the Kcat of PepN containing N259D or Q821E was enhanced with respect to both aminopeptidase and endopeptidase substrates. On the other hand, there was significant decrease in the catalytic activity of the double mutant. Modeling studies demonstrate that the N259-Q821 interaction is located in the vicinity of residues important for catalysis in PepN and specific alterations in this interaction may affect the compactness of the catalytic domain. In summary, this study provides a functional role for the N259-Q821 interaction in modulating the catalytic activity of PepN. Mammalian orthologs of M1 family members play important roles in different physiological processes, e.g. angiogenesis, blood pressure, inflammation, MHC class I antigen presentation etc. PepN is a well characterized M1 family member of microbial origin. The present study on E. Coli PepN provides new knowledge on the roles of: a) distal C-terminal amino acids in repressing exposed hydrophobic amino acids; b) the conserved YL motif during NaSal-induced stress condition; c) the N259 and Q821 interaction in modulating enzymatic activity. The implications of these results on other members of the M1 family are discussed.

Intracellular protein degradation is required for maintaining the cellular proteome and regulating cellular processes. This pathway involves proximal ATP-dependent proteases that unfold and translocate proteins targeted for degradation into catalytic chambers. The large peptides produced are further cleaved by ATP independent endopeptidases, aminopeptidases and carboxypeptidases to release free amino acids. Lon and Clp are the key ATP-dependent proteases in prokaryotes and 26S proteasomes in eukayotes. In general, enzymes involved in the distal processing of peptides are ATP-independent, display greater redundancy and their orthologs are present in most organisms. The aim of the present study was to generate biochemical and functional insights on the ATP-independent enzyme, Peptidase N (PepN), which belongs to the M1 family. Previous studies in our laboratory identified Escherichia Coli PepN, to harbor both amino and endopeptidase activitities. In addition, it is responsible for the cleavage of majority of aminopeptidase substrates in E. Coli and is known to be involved in Sodium salicylate(NaSal)-induced stress. The present study consists of four parts. First, intracellular proteolysis plays an important role for virulence in pathogens. Therefore, it becomes important to study the biochemical properties and roles of enzymes involved in protein degradation. In this direction, a study was initiated to characterize the biochemical properties of Peptidase N from Salmonella enterica serovar Typhimurium(S. typhimurium). To study the contribution of PepN to the overall cystosolic protein degradation in S.typhimurium, a targeted deletion in pepN was generated. Cystosolic lysates of S. typhimurium wild type(WT) and ΔpepN strains were examined for their ability to cleave a panel of aminopeptidase and endopeptidase substrates. The ΔpepN strain displayed greatly reduced cleavage of nine out of a total of thirteen exopeptidase substrates, demonstrating a significant contribution of PepN to cytosolic aminopeptidase activity. S. typhimurium PepN also cleaved the endopeptidase substrate Suc-LLVY-AMC, similar to E. Coli PepN. To understand the physiological role of PepN, WT and ΔpepN were subjected to different stress conditions. During nutritional downshift in combination with high temperature stress, the growth of ΔpepN was significantly reduced compared to WT. Importantly, the PepN overexpressing strains grew better than WT, demonstrating an enhanced ability to overcome this stress combination. The above study clearly underscores the importance of PepN, to play distinct roles during stress. The significance of this study lies in understanding the biochemical and functional properties of a M1 family member from a pathogenic organism. Second, peptidases belonging to the M1 family are widely distributed with orthologs found across different kingdoms. The key amino acids in the catalytic domain are conserved in this family. However, amino acids present in the C-termini are variable and the three available crystal structures of M1 family members display distint differences in organization of this domain. To investigate the functional role of C-termini, progressive deletions were generated in PepN from E.Coli and Tricorn interacting factor F2 from Thermoplasma acidophilum(F2). Catalytic activity was partially reduced inPepN lacking four aa from C-terminus (PepNΔC4) whereas it is greatly reduced in F2 lacking ten amino acids from C-terminus(F2ΔC10) or eleven amino acids from PepN (PepNΔC11). To understand the mechanistic reasons involved, biochemical and biophysical studies were performed on purified WT and C-termini deleted proteins. Increased binding to 8-amino- 1- naphthalene sulphonic acid (ANS) was observed for all C-termini deleted proteins revealing greater numbers of surface exposed hydrophobic amino acids. Further, trypsin sensitivity studies demonstrated that mutant proteins were more sensitive compared to WT. Notably, expression of PepNΔC4, but not PepNΔC11, in E ColiΔpepN increased its ability to resist nutritional and high temperature stress, demonstrating a physiological role for the C-terminus. Together, these studies reveal involvement of distal amino acids in the C-termini of two distant M1 family members in repressing the exposure of apolar residues and enhancing enzyme function. Third, the crystal structure of E. coliPepN displayed the presence of Zn2+. To study the role of metal cofactor, apo-PepN was isolated by chelating the holoenzyme with 1,10-phenanthroline. Among different metals tested, only Zn2+ rescued the greatly reduced catalytic activity of the apo-PepN. Further confirmatory studies were performed using pepN mutants in the conserved GXMEN and HEXXH motifs. No major structural differences were observed in purified mutants(E264A, H297A, and E298A) using circular dichroism (CD) and intrinsic fluorescence studies; however, they lacked catalytic activity. These studies clearly demonstrate that Zn2+ was essential for catalysis but not for the overall structural integrity of PepN. Estimation of the Zn2+ content by atomic absorption spectrometry demonstrated that the WT contained one molecule of zinc per molecule of enzyme. Similar results were obtained in purified proteins of E264A and E298A. residues involved in catalysis. However the Zn2+ amount was greatly reduced in H297A, which is involved in Zn2+ binding. Further, the in vivo role of metal cofactor and catalyis were studied during two established stress conditions. Over expression of the mutants, unlike WT, was unable to rescue the growth of ΔpepN during nutritional down shift and high temperature stress. These results demonstrate that E264, H297 and E298 were required for PepN function during nutritional downshift and high temperature stress. However during NaSal-induced stress condition, overexpression of WT or mutants reduced growth of ΔpepN, demonstrating that PepN function was independent of catalytic activity or metal cofactor. Further studies identified the YL motif, which is conserved in all members of the M1 family, to play a role during NaSal-induced stress. Over expression of Y185F or L186Q did not modulate catalytic activity although growth reduction of ΔpepN in the presence of NaSal was compromised. To understand the mechanisms by which the YL motif plays a role during this condition, Y185F and L186Q mutant proteins were purified. In vitro, both mutant proteins were found to aggregate at a lower temperature and their catalytic activities were more sensitive to temperature, compared to WT. Steady state analysis of WT, Y185F and L186Q were performed to study the modulation of PepN amount during stress conditions. Steady state amounts of Y185F and L186Q mutant proteins were greatly decreased compared to WT, during NaSal-induced stress. Most likely, the lowered amounts of Y185F and L186Q mutant proteins contribute to growth advantage during NaSal-induced stress. Thus, the YL motif in E. Coli PepN reduces protein aggregation and enhances the structural integrity of PepN during selective stress conditions in vivo. In summary, this study clearly identifies metal cofactor and peptidase-dependent and –independent motifs to play distinct functional roles in PepN. Fourth, the crystal structures of known M1 family members have shown that the catalytic domain and mechanism of action are similar. To identify novel residues that may modulate the catalytic activity of PepN, multiple sequence alignment of important M1 family members were performed. The alignment identified a subset of M1 family members, including PepN, containing an aspargine residue which is present two amino acids before glycine in the GAMEN motif. A closer investigation of thecrystal structure of PepN revealed an interaction between N259(Catalytic domain) with Q821 (C-terminal domain). To understand the functional role of this interaction, site-specific mutants were generated: N259D, Q821E and a double mutant, N259D & Q821E. Spectroscopic studies did not reveal any significant differences with respect to global structure or protein stability between purified WT and mutant enzymes. Also, binding to substrates by mutant enzymes was not affected as judged by Km values. However, the Kcat of PepN containing N259D or Q821E was enhanced with respect to both aminopeptidase and endopeptidase substrates. On the other hand, there was significant decrease in the catalytic activity of the double mutant. Modeling studies demonstrate that the N259-Q821 interaction is located in the vicinity of residues important for catalysis in PepN and specific alterations in this interaction may affect the compactness of the catalytic domain. In summary, this study provides a functional role for the N259-Q821 interaction in modulating the catalytic activity of PepN. Mammalian orthologs of M1 family members play important roles in different physiological processes, e.g. angiogenesis, blood pressure, inflammation, MHC class I antigen presentation etc. PepN is a well characterized M1 family member of microbial origin. The present study on E. Coli PepN provides new knowledge on the roles of: a) distal C-terminal amino acids in repressing exposed hydrophobic amino acids; b) the conserved YL motif during NaSal-induced stress condition; c) the N259 and Q821 interaction in modulating enzymatic activity. The implications of these results on other members of the M1 family are discussed.

The cytosolic protein degradation pathway, performed by ATP-dependent proteases and ATP-independent peptidases, plays important roles in several cellular activities, e.g. cell division, cell cycle progression, intracellular signaling, MHC class I antigen presentation, host-pathogen interactions, etc. The roles of ATP-dependent proteases during stress and infection have been studied in great detail but the functional roles of ATP-independent peptidases are not clearly understood. In this study, the functional roles of E. coli or S. typhimurium encoded Peptidase N (PepN), an ATP-independent enzyme belonging to theM1 family of metallopeptidases, were investigated. The thesis will address four different aspects. (i) In the first part, the utility of using E coli ∆pepN to identify and characterize novel peptidases will be shown. It is known that deletion of pepN leads to inability to cleave the majority of in vitro peptidase substrates in E. coli and S. typhimurium. To study the differences between two closely related paralogs of the M17 family, E. coli encoded pepA and pepB were cloned in pBAD24 vector and introduced in E. coli ∆pepN. Peptidase A (PepA) and Peptidase B (PepB) expression increases the cleavage of several aminopeptidase substrates and partially rescues growth of ∆pepN during nutritional downshift and high temperature stress (NDHT), a dual stress involving growth in minimal media at 42°C. Purified PepA and PepB enzymes display broad substrate specificity; however, distinct differences are observed between these two paralogs: PepA is more stable at high temperature whereas PepB displays broader substrate specificity as it cleaves Asp and Insulin B chain peptide. The strategy utilized in this study, i.e. overexpression of peptidases in ∆pepN followed by screening for substrate specificities in total cell extracts, may be used to rapidly identify the substrate preferences of novel peptidases encoded in genomes of different organisms. (ii) The second aspect investigates the functional roles of PepN during stress and infection in S. typhimurium. PepN has two conserved signature motifs of the M1 family, GAMEN and HEXXH, which play roles in substrate recognition and catalysis. To address the roles of catalytic activity of PepN, the residue E-298, which is present in the HEXXH motif and acts as a general base during catalysis, was mutated to A-298 by site-specific mutagenesis and introduced into ∆pepN (pBR322/pepNE298A). Biochemical and biophysical analysis of purified PepN (WT and E298A) revealed loss of catalytic activity of E298A but no major structural changes were observed in comparison to the WT protein. The functional roles of this mutation using ∆pepN expressing pBR322/pepN or pBR322/pepNE298A were investigated using two conditions: (i) Nutritional downshift high temperature (NDHT)stress and (ii) systemic infection in mice. Monitoring growth profiles of different strains demonstrated the requirement of the enzymatic activity of PepN for adaptation and growth to NDHT stress. Earlier studies have shown that S. typhimurium ∆pepN hyper proliferates in peripheral organs during systemic infection in mice. However, expression of wild type (WT)or E298A PepN led to lower colony forming units (CFU), demonstrating that the decrease in CFU is independent of catalytic activity. These observations are consistent with lower serum amounts of inflammatory cytokines, lower tissue damage and increase in survival of mice infected with S. typhimurium expressing WT or E298A PepN. (iii) Although pathogen encoded peptidases are known to be important during infection, their roles in modulating host responses in immunocompromised individuals are not well studied. In the third part of this thesis, the roles of S. typhimurium encoded PepN were studied in mice lacking Interferon-γ (Ifnγ), a cytokine important for immunity. S. typhimurium lacking pepN displays enhanced CFU compared to WT in peripheral organs during systemic infection in C57BL/6 mice. However, Ifnγ-/-mice show higher CFU compared to C57BL/6 mice, resulting in lower fold differences between WT and ∆pepN. Concomitantly, reintroduction of pepN in ∆pepN reduces CFU, demonstrating pepN dependence. In addition, three distinct differences were observed between infection ofC57BL/6 and Ifnγ-/-mice upon infection with different S. typhimurium strains: (i) cytokine profiles, (ii) histological analysis and (iii) mice survival. Overall, the roles of the host encoded Ifnγ during infection with S. typhimurium strains with varying degrees of virulence will be highlighted. (iv) The final aspect of this study reveals differences in gene expression between S. typhimurium grown in rich medium (Luria-Bertani) versus NDHT stress. This adaptation affects several pathways and the gene expression of secretory proteins that are important for virulence in S. typhimurium are greatly reduced during NDHT stress. Also, analysis of secretory protein amounts in different media conditions shows reduction during growth in minimal media plus high temperature stress. The functional consequences of this reduction in secretory protein amounts lead to lower bacterial replication after infection of RAW cells or mice infected via the oral route. In addition, the differences in gene expression between WT and ∆pepN during these conditions were studied. Interestingly, there is reduction in expression of flagellar genes whereas the genes involved in nitrogen metabolism are upregulated in ∆pepN upon exposure to NDHT stress. Further studies were performed by quantifying the motility of different S. typhimurium strains grown in a variety of culture conditions. Overall, this part of the study attempts to compare and contrast the possible adaptive responses of WT and ∆pepN to NDHT stress. Together, this thesis addresses multiple aspects of the biochemistry and roles of the enigmatic PepN during stress and infection.

The cytosolic protein degradation pathway, performed by ATP-dependent proteases and ATP-independent peptidases, plays important roles in several cellular activities, e.g. cell division, cell cycle progression, intracellular signaling, MHC class I antigen presentation, host-pathogen interactions, etc. The roles of ATP-dependent proteases during stress and infection have been studied in great detail but the functional roles of ATP-independent peptidases are not clearly understood. In this study, the functional roles of E. coli or S. typhimurium encoded Peptidase N (PepN), an ATP-independent enzyme belonging to theM1 family of metallopeptidases, were investigated. The thesis will address four different aspects. (i) In the first part, the utility of using E coli ∆pepN to identify and characterize novel peptidases will be shown. It is known that deletion of pepN leads to inability to cleave the majority of in vitro peptidase substrates in E. coli and S. typhimurium. To study the differences between two closely related paralogs of the M17 family, E. coli encoded pepA and pepB were cloned in pBAD24 vector and introduced in E. coli ∆pepN. Peptidase A (PepA) and Peptidase B (PepB) expression increases the cleavage of several aminopeptidase substrates and partially rescues growth of ∆pepN during nutritional downshift and high temperature stress (NDHT), a dual stress involving growth in minimal media at 42°C. Purified PepA and PepB enzymes display broad substrate specificity; however, distinct differences are observed between these two paralogs: PepA is more stable at high temperature whereas PepB displays broader substrate specificity as it cleaves Asp and Insulin B chain peptide. The strategy utilized in this study, i.e. overexpression of peptidases in ∆pepN followed by screening for substrate specificities in total cell extracts, may be used to rapidly identify the substrate preferences of novel peptidases encoded in genomes of different organisms. (ii) The second aspect investigates the functional roles of PepN during stress and infection in S. typhimurium. PepN has two conserved signature motifs of the M1 family, GAMEN and HEXXH, which play roles in substrate recognition and catalysis. To address the roles of catalytic activity of PepN, the residue E-298, which is present in the HEXXH motif and acts as a general base during catalysis, was mutated to A-298 by site-specific mutagenesis and introduced into ∆pepN (pBR322/pepNE298A). Biochemical and biophysical analysis of purified PepN (WT and E298A) revealed loss of catalytic activity of E298A but no major structural changes were observed in comparison to the WT protein. The functional roles of this mutation using ∆pepN expressing pBR322/pepN or pBR322/pepNE298A were investigated using two conditions: (i) Nutritional downshift high temperature (NDHT)stress and (ii) systemic infection in mice. Monitoring growth profiles of different strains demonstrated the requirement of the enzymatic activity of PepN for adaptation and growth to NDHT stress. Earlier studies have shown that S. typhimurium ∆pepN hyper proliferates in peripheral organs during systemic infection in mice. However, expression of wild type (WT)or E298A PepN led to lower colony forming units (CFU), demonstrating that the decrease in CFU is independent of catalytic activity. These observations are consistent with lower serum amounts of inflammatory cytokines, lower tissue damage and increase in survival of mice infected with S. typhimurium expressing WT or E298A PepN. (iii) Although pathogen encoded peptidases are known to be important during infection, their roles in modulating host responses in immunocompromised individuals are not well studied. In the third part of this thesis, the roles of S. typhimurium encoded PepN were studied in mice lacking Interferon-γ (Ifnγ), a cytokine important for immunity. S. typhimurium lacking pepN displays enhanced CFU compared to WT in peripheral organs during systemic infection in C57BL/6 mice. However, Ifnγ-/-mice show higher CFU compared to C57BL/6 mice, resulting in lower fold differences between WT and ∆pepN. Concomitantly, reintroduction of pepN in ∆pepN reduces CFU, demonstrating pepN dependence. In addition, three distinct differences were observed between infection ofC57BL/6 and Ifnγ-/-mice upon infection with different S. typhimurium strains: (i) cytokine profiles, (ii) histological analysis and (iii) mice survival. Overall, the roles of the host encoded Ifnγ during infection with S. typhimurium strains with varying degrees of virulence will be highlighted. (iv) The final aspect of this study reveals differences in gene expression between S. typhimurium grown in rich medium (Luria-Bertani) versus NDHT stress. This adaptation affects several pathways and the gene expression of secretory proteins that are important for virulence in S. typhimurium are greatly reduced during NDHT stress. Also, analysis of secretory protein amounts in different media conditions shows reduction during growth in minimal media plus high temperature stress. The functional consequences of this reduction in secretory protein amounts lead to lower bacterial replication after infection of RAW cells or mice infected via the oral route. In addition, the differences in gene expression between WT and ∆pepN during these conditions were studied. Interestingly, there is reduction in expression of flagellar genes whereas the genes involved in nitrogen metabolism are upregulated in ∆pepN upon exposure to NDHT stress. Further studies were performed by quantifying the motility of different S. typhimurium strains grown in a variety of culture conditions. Overall, this part of the study attempts to compare and contrast the possible adaptive responses of WT and ∆pepN to NDHT stress. Together, this thesis addresses multiple aspects of the biochemistry and roles of the enigmatic PepN during stress and infection.

Nowadays, in a world where competion for market presence is fearless, innovating becomes essential for companies to survive and thrive. Given that the majority of companies worldwide a family business, it is relevant to understand how do these businesses act with regards to innovation. For many family business worldwide innovation is a distinction factor, that allows them to continue to exist throught different generations. In this context, the main purpose of the present dissertation is to understand to what extent are in fact family businesses more innovative than the other kind of enterprises and their capacity to innovate. For that purpose the literature review will, on a first stage address the family business’ relation with innovation, analysing different positions in literature on both the willingness and the abilty of these type of companies to innovate. On a second stage, a study on a family business – the GL company – is presented, in order to provide a practical example of an innovative family business. For the purpose of presenting an internal perspective on the company’s posture towards innovation, interviews were conducted to two senior family members - the CEO and a manager. The qualitative analysis of the interviews has provided practical insights on how GL behaves towards innovation, by applying the key concepts exposed on the literature review to the company’s reality. The theorectical aspects of the present thesis combined with GL’s case study intended to provide a comprehensive approach to the issues relating to the innovation processes within family businesses.
Atualmente, num mundo onde a competição pela presença no mercado é destemida, a inovação torna-se essencial para as empresas sobreviverem e prosperarem. Dado que a maioria das empresas em todo o mundo são familiares, é importante entender como as mesmas agem no que diz respeito à inovação Para muitas empresas familiares em todo o mundo, a inovação é um fator de distinção, que lhes permite perlongar por várias gerações. Neste contexto, o principal objetivo da presente dissertação é entender até que ponto as empresas familiares são de fato mais inovadoras do que o outro tipo de empresa e a sua capacidade de inovar. Para esse fim, a revisão de literatura, num primeiro estágio, abordará a relação da empresa familiar com a inovação, analisando diferentes posições na literatura sobre a vontade e a capacidade deste tipo de empresa inovar. Num segundo estágio, é apresentado o estudo sobre uma empresa familiar - a empresa GL - para fornecer um exemplo prático de uma empresa familiar inovadora. Com o objetivo de apresentar uma perspetiva interna da postura da empresa em relação à inovação, foram realizadas entrevistas a dois membros da família de gerações diferentes. A análise qualitativa das entrevistas forneceu informações práticas sobre como a GL se comporta relativamente à inovação, aplicando os conceitos-chave expostos na revisão de literatura à realidade da empresa. Os aspetos teóricos da presente tese combinados com o estudo de caso da GL pretendem fornecer uma abordagem abrangente para as questões relacionadas aos processos de inovação nas empresas familiares.

This study aims to produce an analysis of the universe of associates belonging to the Portuguese Association of Family Firms. The goal is not only to characterize them, but also to establish comparisons between them and the Portuguese macroeconomical scenario. The beginning of this study is an introduction to the main topics surrounding the scope of family businesses that are currently being investigated in the academia. In order to complete the framing of the subject and the produced analysis, a summary of what has been debated on this topic was made, emphasizing the concerns, challenges and tendencies that this type of companies has felt. The confrontation between these topics and the results obtained will be the target of the final discussion. It should be noted that the results and analyzes produced were based on data from 464 companies. That data was collected with the goal of producing indicators capable of allowing a descriptive analysis of the firms and also the macroeconomic impact they have on a national level. In order to comply with the goals of the study, the indicators generated were produced to provide an image that characterizes the sectors of activity as well as the legal form, geographical location, size and performance for the firms in study. The data covers the time period that goes from 2010 to 2016. Finally, in terms of macroeconomic impact, the analysis carried by this study focus on topics such as Corporate Income Tax, Personnel Costs and Gross Domestic Product.
Com este estudo pretende-se realizar uma análise ao universo de associados da Associação das Empresas Familiares de forma a caracterizá-los e estabelecer algumas comparações com o panorama nacional português. No entanto, surge como principal objetivo do estudo, a elaboração de alguns indicadores que ajudem a revelar o impacto que este universo tem no contexto nacional. A abordagem inicial ao tema é iniciada com uma análise aos principais temas em debate no universo académico e de investigação relativamente à temática das empresas familiares e do crescente interesse no estudo da mesma. De forma a completar o enquadramento do tema e as análises produzidas, foi feita uma recolha daquilo que vem sendo debatido, dando enfase ás preocupações, desafios e tendências que este tipo de empresas tem sentido. O confronto entre esses tópicos e os resultados obtidos será o grande alvo de discussão final. Importa referir que os resultados e análises produzidas foram construídas com base em dados de 464 empresas com o objetivo de produzir indicadores que permitissem obter uma análise descritiva mas também de impacto macroeconómico. Assim, foram apresentados valores referentes aos anos de 2010 até 2016, que permitissem obter uma imagem caracterizadora dos sectores de atividade, forma jurídica, localização geográfica, dimensão e performance, das empresas em estudo. A nível do impacto macroeconómico as análises visam tópicos como o Imposto sobre o Rendimento das Pessoas Coletivas, Custos com Pessoal e o Produto Interno Bruto.

A presente dissertação aborda a problemática da sucessão nas empresas familiares e o impacto que as notícias divulgadas na comunicação social, durante o processo de sucessão, têm no valor da empresa. A sucessão é referida, por muitos autores como o maior desafio enfrentado pelas empresas familiares (Handler, 1994). Estatísticas reportam que 65 a 80% das empresas ao nível global têm cariz familiar (Ussman, 2004) e que apenas 30% das empresas familiares chegam à segunda geração e 10% à terceira (Beckhard & Dyer, 1983). Tendo como ponto de partida a conjetura que a notícia da sucessão facilmente cria instabilidade envolta dos principais stakeholders: sejam acionistas, fornecedores, clientes ou empregados, entre outros, esta dissertação analisa o efeito, positivo/negativo, que as notícias que saíram na comunicação social sobre a sucessão de um grupo empresarial familiar, nomeadamente, o Grupo Jerónimo Martins, tiveram no valor de mercado da empresa, isto é, no valor da ação. A relevância desta abordagem é justificada pela facilidade e rapidez de acesso à informação que atualmente o mercado dispõe e que muitas vezes a empresa não consegue controlar. Nesta era da informação e da tecnologia, é fundamental que as empresas mantenham o mercado informado sobre como está a ser desenvolvido o processo de sucessão da empresa, de modo a evitar que especulações levantem instabilidade à empresa.
This thesis addresses the problematic of succession in family businesses and the effect of the media has, during the succession period, on the company value. The succession is referred, by some authors as the biggest challenge faced by family businesses (Handler, 1994). Statistics show that 65 to 80% of companies, globally, are family-oriented (Ussman, 2004) and that only 30% of these companies survive into the second generation and only 10% into the third (Beckhard & Dyer, 1983). Assuming that the effect created by the news of the succession easily creates instability around the main stakeholders: shareholders, suppliers, customers or employees, among others, this thesis analyses the effect, positive or negative, that the news published by the media about the succession on a family-oriented business group, mainly, Grupo Jerónimo Martins, had on the company’s market value, that is, the share value. The relevance of this approach is justified by the ease and speed of access to information than the market currently provides and that the company can’t always control. In this information and technology era, it’s essential that the companies maintain the marker informed about how it is being handled the company’s process of succession, so that speculations don’t bring instability to the company.

Among the fossilized remains of early Eocene mammals collected from the Buck Spring Quarries of Wyoming are the dentitions of several previously undescribed nyctitheriids. Comparison of this material (from the Lost Cabin Member of the Wind River Formation, late Wasatchian Land Mammal Age (LMA), Lost cabinian Land Mammal Subage (LMSA, Wa-7)) to closely related taxa requires the description of a new genus and species of nyctitheres, Acrodentis rosenorum, as well as a new species of Nyctitherium, N. krishtalkai. A. rosenorumis similar to closely related Nyctitherium and Leptacodon, but is set apart by its distinctively shaped anterodorsally curving paraconid, together with a protoconid and metaconid that project away from one another forming an open trigonid. In the upper teeth, the paracone and metacone also project in slightly different directions, suggesting the association of upper and lower molar morphology. The protocone is nearly centered laterally between the paracone and metacone, unlike Leptacodon, and the hypoconal shelf is less broadly expanded than in Nyctitherium. N. krishtalkai, though similar to N. velox and N. serotinum, differs from these species in that the cristid obliqua terminates where it strikes the postvallid, the hypoconulid does not as closely twin the entoconid, and the entoconid occurs slightly higher than the hypoconid. In the upper teeth, the conules and conular wings are more developed than in previously described species, the hypocones, though broadly expanded into shelves on M1–2, are less developed, and the paracone and metacone of M3are less reduced. The evolutionary context of these two new groups was investigated with a cladistic analysis based on dental characters, including species from the described genera Nyctitherium, Leptacodon, Plagioctenodon, Plagioctenoides, Pontifactor, Wyonycteris, and Lima­conyssus. Palaeictopsspp. served as the outgroup. The results of this analysis suggest a close relationship between Acrodentis, Leptacodon, and Nyctitherium, in which A. rosenorumappears closely related to an ancestor intermediate between Leptacodon and Nycti­therium. N. krishtalkaiis the most primitive of its genus and is the most closely related to A. rosenorum. Whereas a cladistic analysis involving only nyctitheres should not be used as basis to divide the family, two major clades within the Nyctitheriidae of North America may exist: one including the genera Leptacodon, Plagioctenodon, Nyctitherium, and Acrodentisand the other including Wyonycteris, Limaconyssus, and “Plagioctenoides.”

Abstract non-zero angular momentum ensures that a d electron never approaches very close to the nucleus: the repulsive centrifugal force of the motion rises more rapidly than the attractive Coulombic force, and the net force is repulsive as r approaches zero. The boundary surfaces of the five real forms of the wave functions of the 3d orbitals are shown in Fig. D.1 (the boundary surfaces of other d orbitals are similar). All the orbitals have two nodal planes. The peculiar shape of the d22 orbital is a result of forming linear combinetions of the °complex forms of the orbitals in order to depict them more simply. Figure D.2 indicates how the five orbitals look when represented as complex wave functions, which is appropriate when we want to identify each orbital with a specific value of m1: now they all look like members of the same family.

This paper presents the details of a compact embedded-computing module designed to meet a variety of pedagogical objectives within mechatronics, controls, and robotics. Built around an ATmega32U4 microcontroller, the 1.8 × 4.0 centimeter module has flash memory for program and data storage, 25 general-purpose input/output lines, four timer/counters, 12 channels of 10-bit analog-to-digital conversion, and support for a variety of serial communications protocols, including USB. The unit adapts easily to a solderless breadboard for quick prototyping, and requires only an external 5-volt power source for operation. Furthermore, it can be programmed directly over a USB connection to a computer, thereby eliminating the need for a separate programming device. As a member of the AVR family of microcontrollers, the development tools for the processor are freely available for Windows, Mac, and Linux. When assembled in sufficient quantity, the part cost for each module is approaching $10US, making it a low-cost solution for a variety of tasks. To enable students and professors to explore both the module and the host of application principles, we have chosen to post the design files and documentation on a publicly-accessible wiki, leaving room for collaborative improvements and the sharing of technology with other educational institutions.

Context: Recently, with the advance of neuroimaging modalities, the windows of reperfusion therapy in patients with acute stroke have been reviewed and extended, especially for mechanical thrombectomy. Case report: 81 year old patient, previously hypertensive and dyslipidemic, fully functional (modified Rankin scale = 0), admitted to the emergency room of a tertiary hospital with global aphasia, right hemiparesis, right homonymous hemianopsia and severe hypoesthesia of the right upper limb, scoring 26 on the NIHSS, with report of having contacted family members for the last time 15 hours before admission. She was treated according to the institution’s acute stroke protocol, and underwent non-contrast brain computed tomography (CT), perfusion CT with Rapid CT protocol and cerebral artery + neck angio-CT, which ruled out bleeding and showed an ASPECTS of 8, an estimated ischemic core volume of 17 mL, and an area with hypoperfusion of 118 mL (perfusional mismatch of 101 mL), besides occlusion of the M1 segment of the left middle cerebral artery. Thus, she was submitted to chemical thrombolysis, with a decrease in NIHSS score to 15 and evolving without complications upon hospitalization. Conclusions: In patients with uncertain ictus, the use of advanced neuroimaging modalities, such as perfusion tomography with Rapid CT protocol, may assist in the indication of reperfusion therapies safely.

Aero-derivative gas turbines have been successfully serving the power generation, mechanical drive, and marine markets for 40 years. These products are well suited for distributed generation, with sizes in the range from 3 MW to 50+ MW. The Rolls-Royce group of companies provide vertical integration for aero-derivative based energy systems, having marketing, sales, manufacturing, packaging, distribution, and customer service capabilities. The 3– 6 MW, 501-K family serves power generation and cogeneration applications. The new 6–8 MW 601 is used for cogeneration and mechanical drive. The 15 MW Avon is widely applied to mechanical drives, offering exceptional reliability and low life cycle cost. The RB211 provides over 30 MW at high efficiency, and is used in mechanical drive and electrical generation. The 42% efficient, 50 MW, Trent is primarily intended for electrical generation. This engine retains a higher than usual degree of commonality with aero production modules, thus retaining the cost advantage of high volume production and benefits from continuous improvements in aero engines. Plans: Cost reduction of mature existing products will be achieved by “industrialization”, e.g. by alloy changes and shape simplification, of parts no longer in aero production. Better integrated packaging and “more electric aircraft” features are rapidly becoming a necessity in the competitive marketplace. The trend is toward minimizing and possibly eliminating mechanical drives and other components in a gas turbine to improve product quality, efficiency, reduce product cost, while enhancing product quality and the environment. In this regard, the approach being taken near term is to substitute normal oil bearings with Active Magnetic Bearings. Such an action would help eliminate high cost skid lubrication system components and some environmental hazards as well as reducing maintenance. Several programs will make contributions to environmental improvements through reduced emissions and the use of “renewable” fuels. A prototype 501-K has been supplied to operate on gasified coal, a reduced emissions path to generating electricity from coal. A dual fuel DLE combustion system for very high pressure ratio and turbine temperature is in development for the Trent, having downward compatibility with other company products. The Next Generation Gas Turbine (NGGT) project, sponsored by the US Department of Energy, will use an existing engine core. Advanced modules, including a long life “spiral” recuperator and cycle enhancements combine to yield 50% cycle efficiency at a reduced cost per kW. The goal is to produce a 50 MW class plant with “combined cycle efficiency at simple cycle cost.” The NGGT is suited to using alternate fuel for part of the energy input. Following evaluation of fuel cell/gas turbine hybrids, a specially suited gas turbine development is being initiated with sponsorship by the U.S. Department of Energy. The company is also conducting a solid oxide fuel cell program. An auxiliary power unit(APU) was developed and is now in production for the M1 tank. A “microturbine” derivative of this product is being considered for distributed generation.