Dissertations / Theses on the topic 'Gefsoc'

2009/2010
I suoli globalmente contengono circa 2500 Pg di C in forma minerale ed organica (ca 1550 Pg) ed il flusso annuo da e verso l’atmosfera – che attualmente contiene circa 760 Pg C – coinvolge circa 120 Pg di C. Questi scambi di carbonio sono clima-dipendenti; l’effetto del cambiamento climatico sui depositi di carbonio del suolo è perciò di importanza critica, in quanto anche piccole variazioni di un deposito di tale entità possono determinare importanti conseguenze sulla concentrazione di anidride carbonica in atmosfera, innescando meccanismi retroattivi positivi. Per fare delle previsioni sui cambiamenti dei depositi di carbonio per effetto dei cambiamenti climatici o di altre variabili è neccesario ricorrere a dei modelli; in particolare, per le stime a scala nazionale e regionale si impiegano dei modelli che operano a livello di ecosistema e che vengono abbinati a dei SIT. Vista la forte sinergia con la mitigazione e adattamento ai cambiamenti climatici, la ricerca è stata volta a stimare il potenziale di sequestro e accumulo di sostanza organica nei terreni agricoli del Veneto, con un occhio di riguardo per la gestione sostenibile. In secondo luogo, a fornire uno strumento per la contabilizzazione del sequestro di carbonio nei sistemi agricoli. Si è individuato nel GEFSOC Modelling Sistem uno strumento idoneo per il perseguimento di questi obiettivi; il GEFSOC MS integra due modelli meccanicistici ampiamente sperimentati – Century e RothC – ed il metodo empirico dell’IPCC e li interfaccia con un SOTER-DB e con un GIS. L’uso e la gestione del suolo ed i loro cambiamenti sono variabili fondamentali nel determinare il contenuto di carbonio nei depositi del terreno; poiché manifestano i loro effetti anche secoli dopo che si sono verificati, è necessario ricostruire la loro storia almeno negli ultimi 100 anni. Nel corso della ricerca è stato ideato e sperimentato per la prima volta in questo settore un approccio spazialmente esplicito alle transizioni di uso e gestione del suolo, utilizzando materiale cartografico di varia natura e completando la raccolta dati con statistiche agrarie e fonti storiche. Le simulazioni sono state fatte contemplando due diversi scenari di cambiamento climatico (PCM-B1 e Had3A1FI) spinti fino al 2100. L’analisi dei risultati prodotti evidenzia che i terreni più ricchi in carbonio sono maggiormente soggetti a perdite, mentre quelli poveri, anche se in misura modesta, incrementano il loro contenuto; la tendenza, quindi, è di avvicinarsi ad una maggiore omogeneità. Per quel che riguarda gli usi del suolo, seminativi ed aree agricole eterogenee sono le categorie che hanno manifestato tassi di incremento superiori. I tassi di variazione, comunque, sono tendenzialmente in calo e, per lo scenario di cambiamento climatico meno marcato, ad un certo punto (2070 ca) si portano su valori negativi: questo fatto si ripercuote sui depositi del terreno, che complessivamente mostrano un incremento nel lungo periodo, ma via via più contenuto, fino a raggiungere un massimo e quindi cominciare ad emettere negli ultimi decenni della simulazione. I depositi dei terreni sottoposti allo scenario di cambiamento più marcato, invece, non hanno subito flessioni ed hanno garantito, anche se in misura calante, il sequestro lungo tutto il periodo della simulazione. Questo dato può essere di qualche interesse nello studio degli effetti della temperatura sul rapporto produzione di biomassa-accumulo/decomposizione. L’analisi delle mappe dei depositi e dei tassi di variazione annua ad ettaro prodotte con la sperimentazione, ed il confronto delle stesse con carte del contenuto del carbonio nei terreni di doversa origine e natura, rivelano l’utilità dell’approccio spazialmente esplicito nella definizione delle transizioni dell’uso e gestione del suolo; è possibile infatti riconoscere, dalla zonizzazione, i tematismi che possono avere avuto un peso preponderante nel determinare peculiari situazioni; questo consente di indagarle, verificare la validità delle assunzioni fatte in fase di progettazione, modificare eventualmente la parametrizzazione e reindirizzare le simulazioni. Ad una livello di risoluzione comparabile a quello degli strati informativi di partenza. Alla luce della sperimentazione effettuata, si ritiene che il GEFSOC Modelling System meriti grande considerazione per quanto attiene la contabilizzazione del carbonio nei sistemi agricoli – problematica inevasa fino ad oggi in Italia; quale strumento a supporto del processo decisionale; per le possibili sinergie nella pianificazione di campionamenti e sperimentazioni attinenti; per l’approfondimento della ricerca nell’ambito delle relazioni tra clima e suolo.
XXIII Ciclo
1970

My graduation work is focused on logistics aplication at specific provider of logistics services. In theoretical part I describe development of logistics, types and links of supply chain and supply chain in automotive industry. In my application part I describe company GEFCO and car producer TPCA. I analyse present situation, standard and non-standard activities solved by department GEFCO Overland. Next I appreciate questionnaire survey, where I find out hot company's problems and try to give their solution that can increase effectivity, decrease costs and provide more valuable services to company's customers.

The thesis is focused on the evaluation of the financial performance of the company GEFOS, Inc. The aim is to evaluate the financial performance and impact of the original recommendations, which the company has applied, from the previous bachelor thesis. Based on the findings, a successive financial development will be assessed together with further possible measures for economic growth of the company. The first part contains theoretical knowledge of the apparatus of a financial analysis and selected methods and analysis of macro environment. In the second part, which is based on the stated goal, the gained expertise is used in practice. The work also contains, based on the results of the analysis, a sectoral comparison that helps to illustrate the overall financial situation of the selected company.

My master thesis deals with the processes of empty packaging in GEFCO company. The goal is to analyze these processes and propose solutions that the company would bring cost savings and optimization of the flows. In the theoretical part I define returnable packaging, their functions, types, trends and compare different logistic providers. The practical part is about GEFCO focusing on the Czech branch and its processes with returnable packaging

The goal of this thesis is to assess benefits of implemented quality management system in GEFCO ČESKÁ REPUBLIKA s.r.o. and to propose possibilities of its further improvements by analysis of data provided by the company. The work starts with the explanation of its theoretical background, methods and conclusions of the three reference studies then follow an overview of company's quality management system, trends in selected indicators and the identification of tangible benefits of implementation using a questionnaire for comprehensive self-assessment according to ISO 10014. The ISO certification has had provably positive impacts, in the frame of a positive economic trend, particularly in the areas of customer focus and continual improvement. In the end author suggests, among other proposals for improvement, mainly to implement sophisticated intranet application and Balanced Scorecard.

The method of combination p-values from multiple tests is the foundation for some studies like meta-analysis and detection of signal. There are tremendous methods have been developed and applied like minimum p-values, Cauchy Combination, goodness-of-fit combination and Fisher’s combination. In this paper, I tested their ability to detect signals which is related to real case in biology to find out significant single-nucleotide polymorphisms (SNPs). I simulated p-values for SNPs logistics regression model and test 7 combination methods’ power performance in different setting conditions. I compared sparse or dense signals, dependent or independent and combine them in gene-level or pathway-level. One method based on Fisher’s combination called Omni-TFisher is ideal for most of the situations. Recent years, genome-wide association studies (GWASs) focused on BMD-related SNPs at gene significance level. In this paper I used Omni-TFisher to analyses real data on haplotype blocks. As a result, haplotype blocks can find more SNPs in non-coding and intergeneric regions than gene-based and save computational complexity. It finds out not only known genes, but also other genes need further verification.

The aim of this study has been to investigate in feminist terms whether or not the character Grendel’s mother symbolizes early matrilineal tribes in the form of the Norse goddess Gefion, also claimed to be the Earth goddess. The claim has been brought forward in an article by Frank Battaglia on the grounds that the chthonic deity is mentioned on several occasions in Beowulf. However, Grendel’s mother’s possible connection to the goddess has not been treated extensively in a feminist context, despite the apparent link between feminism and matrilineal tribes in a patriarchal hierarchy. The modern translations of her character as a monster stand in stark contrast to the original manuscript where she is depicted as an aglӕcwif, “female warrior”. The subject has given rise to a number of feminist researches on the theme of the so called “woman-as-monster” stereotype. These argue that Grendel’s mother has fallen victim to enforced marginalization due to etymological faults as well as sexist stereotypes in Anglo-Saxon literary culture. On the background of Moi’s definition of a woman and Kristeva’s concept of the abject, results demonstrate that Grendel’s mother may very well symbolize the female Other in a new social order, embodied or represented as the Earth goddess.

RasGRP1 is an intracellular signaling protein expressed in lymphocytes that is responsible for activating Ras GTPases. Positive regulation of RasGRP 1 requires translocation to cellular membranes where lipid-anchored Ras can be accessed. Plasma membrane localization of RasGRP 1 in response to antigen receptors requires both the Cl domain and the plasma-membrane targeting (PT) domain. The Cl domain binds to diacylglycerol (DAG) at membranes. The PT domain binds its putative ligand at the plasma membrane and is negatively regulated by an adjacent suppressor of PT (SuPT) domain. RasGRP1 also contains a pair of EF-hands, with Ca²⁺-binding capability, but with no known regulatory role. In DT4O cells, RasGRP1 translocates to the plasma membrane and activates the Ras ERK pathway in response to B cell receptor (BCR) signaling. By introducing point mutations in the Ca²⁺-binding loops of each of the EF-hands, I found that a potential Ca²⁺- interaction loop in the first EF-hand is required for RasGRP1 translocation and the consequential activation of the Ras-ERK pathway in response to BCR signaling. However, RasGRP1 translocation is not regulated by BCR-generated Ca²⁺ flux. EF-hands were not required for Cl domain-mediated membrane localization, but were needed for PT-mediated plasma membrane targeting. EF-hands enhanced PT-domain mediated plasma membrane localization by repressing the SuPT domain. The REM and GEF domains, which co ordinately bind to and catalyze guanine nucleotide exchange on Ras GTPases, needed to be present and Ras-bound for this EF-hand mechanism to be effective. When not bound to Ras, the REM-GEF domain complex suppressed both plasma membrane and endomembrane targeting of RasGRP 1 by an EF-hand independent mechanism. Finally, membrane localization and activation of a naturally occurring splice variant of RasGRP 1, found overexpressed in systemic lupus erythematosus (SEE) patients, was examined. This splice variant lacks exon 11, which encodes the segment of RasGRP1 between the GEF domain and the first EF-hand. Removal of exon 11 resulted in a defect in plasma membrane localization that was partially overridden by deletion of SuPT, while membrane localization control via the REM-GEF complex was not affected. Therefore, exon 11 deletion via alternative splicing appears to functionally disable the first EF-hand of RasGRP1.

Diplomová práce se zaměřuje na oblast ?inbound? logistického řetězce v automobilovémprůmyslu. Charakterizuje roli logistického integrátora a jeho funkce při zásobování výrobního závodu. Na konkrétním příkladu firmy GEFCO jako výhradního poskytovatele logistických služeb pro klienta PSA Peugeot Citroën práce ukazuje možnosti zkvalitnění služeb logistického integrátora. Práce vychází z roční odborné praxe uskutečněné autorkou v sídle společnosti GEFCO v Paříži v období červenec 2006- červen 2007.

GABAA and glycine receptors are clustered at inhibitory synapses via interactions with the scaffolding protein gephyrin. In turn, gephyrin is translocated to synapses by collybistin, a guanine nucleotide exchange factor (GEF) for Cdc42. Mutations in the human collybistin gene (ARHGEF9) give rise to a range of symptoms including anxiety, epilepsy and intellectual disability. However collybistin knockout mice revealed a selected loss of clustering of distinct GABAA receptor subtypes, whilst glycine receptors clustering remained intact. This suggests that other GEFs might also contribute to gephyrin and inhibitory receptor clustering. This thesis describes the identification and charactertisation of two novel GEFs that were indentified in a yeast two-hybrid screen using a gephyrin bait: IQSEC2 and IQSEC3. Full-length IQSEC2 did not interact with gephyrin in vitro, and is located at excitatory synapses in vivo, so is unlikely to have a role in GABAAR and GlyR clustering. However, four missense mutations in IQSEC2 were found in families with X-linked intellectual disability (XLID) that impair either calmodulin binding to the IQ-like domain (R359C) or ArfGEF activity (R758Q, Q801P and R863W). By contrast, full-length IQSEC3 did interact with gephyrin in vitro and co-localised with gephyrin and inhibitory receptors in vivo, making this ArfGEF a plausible clustering factor. I also show that a gene fusion affecting IQSEC3 may also result in autosomal intellectual disability associated with behavioural defects. Lastly, I examined the interactions between inhibitory GABAAR and GlyR subunits with gephyrin, mapping binding sites for gephyrin on the GABAAR α3 subunit. My analysis revealed that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABAAR α3 M3-M4 domain and the motif SMDKAFITVL at the N-terminus of the gephyrin E domain. Site-directed mutagenesis of the gephyrin E-domain revealed that GABAAR α3 and GlyR β subunits bind to an overlapping site on the gephyrin E-domain.

La famille des récepteurs couplés aux protéines G (GPCRs) est la plus grande famille de récepteurs membranaires et est très importante au niveau de la pharmacologie, de la physiologie et des industries pharmaceutiques. En effet, les GPCRs composent 1 à 2% du génome des mammifères et environ 60% des médicaments sur le marché ciblent des GPCRs. Dans notre laboratoire, nos études portent principalement sur les récepteurs du thromboxane A[indice inférieur 2] (TP). On dénombre deux isoformes de TP, soit TP[alpha] et TP[bêta]. Nous avons déjà démontré que seule TP[bêta] est capable d'internalisation induite par agoniste et constitutive. Le récepteur TP[bêta] signale principalemement via la protéine G hétérotrimérique G[indice inférieur q]. Dernièrement, nous avons observé qu'ARNO, une GEF d'ARF6, interagit avec la protéine G[indice inférieur q] et est impliquée dans l'endocytose de TP[bêta]. Dans cette présente étude, nous démontrons que plusieurs GEFs d'ARF6 interagissent avec G[indice inférieur q]. Les GEFs sont responsables de l'échange du GDP en GTP et permettent l'activation de leurs protéines cibles. Ainsi, nous démontrons que les GEFs GRP1, ARF-GEP[indice inférieur 100], cytohesine-1 et EFA6 co-immunoprecipitent avec G[indice inférieur q]. Dans le cas de GRP1 et cytohesine-1, l'état d'activation de la sous-unité [alpha] de G[indice inférieur q] est important. En effet, GRP1 se lie à G[indice inférieur q] uniquement lorsqu'elle est sous sa forme active, c'est-à-dire liée au GTP. Pour ce qui est de cytohesine-1, cette GEF se lie au forme active et inactive de G[indice inférieur q], mais plus fortement à la forme active. D'un autre côté, ARF-GEP[indice inférieur 100] et EFA6 se lient autant à G[indice inférieur q]-GDP qu'à G[indice inférieur q]-GTP. Par la suite, nous nous sommes concentrés sur l'interaction cytohesine-1 - G[indice inférieur q]. Nous avons démontré que cytohesine-1 lie directement G[indice inférieur [alpha]q] et que la portion N-terminale de cytohesine-1 (domaines CC et Sec7) est responsable de cette interaction. Par la suite, nous avons étudié l'effet de cytohesine-1 sur l'internalisation de TP[bêta]. Dans cette portion de l'étude, nous utilisons le mutant cytohesine-1(E157K) qui est un mutant de cytohesine-1 qui ne possède plus d'activité GEF. Nous démontrons que cytohesine-1 et cytohesine-1(E157K) augmente et diminue respectivement l'expression de surface de TP[bêta] dans les cellules HEK293. De plus, la surexpression du mutant cytohesine-1(E157K) a un effet négatif sur l'internalisation de TP[bêta]. Les cellules surexprimant cytohesine-1(E157K) ont 50% moins d'endocytose de TP[bêta] après 60 minutes de stimulation avec le mimétique du TXA[indice inférieur 2], le U46619. Aussi, l'immunofluorescence a permis de visualiser que le mutant cytohesine-1(E157K) a un effet drastique sur la morphologie des cellules HEK293. En bref, nos résultats démontrent pour la première fois que plusieurs GEFs d'ARF6 interagissent avec G[indice inférieur q]. Aussi, cytohesine-1 se lie directement à G[indice inférieur [alpha]q] via sa portion N-terminale et son activité GEF semble impliquée au niveau de la réorganisation du cytosquelette et de l'endocytose de TP[bêta].

Transport is considered a crucial area of a Supply Chain since it is responsible for bonding all the agents that are part of it. To obtain maximum profit in distribution, it is necessary to optimize 3 variables which are referred as the vertices of the Logistics Triangle: Cost, Time and Quality, which means that products must arrive at the final customer at the right time, with the minimum cost for the company and with the maximum quality for the customers. Routing optimization is one of the most complex areas to apply since there are a lot of variables that cannot be measured and controlled, depending on each scenario. This project was developed to study how could this company reach the maximum quality of the service while influenced by external and internal factors which influence directly the distribution of orders placed by customers, along with restrictions associated to each scenario. Therefore, the main objective is to optimize several routes of distribution that daily supply the customers of the company. This project is inserted in the Vehicle Routing Problems (VRP) and started by, firstly, understand the business of the company and then collecting various types of data which were treated and analysed by using Microsoft Excel and its functionalities, such as with formulas and Pivot Tables. Solutions and scenarios were analysed and presented using an Excel Spreadsheet (VRP Spreadsheet).
O Transporte é considerado uma parte crucial de qualquer Cadeia de Abastecimento, visto ser responsável por interligar todos os agentes que fazem parte da mesma. Para obter máximo proveito na distribuição, é necessário otimizar as 3 variáveis que são referidas como os vértices do Triângulo Logístico: Custo, Tempo e Qualidade, o que significa que os produtos têm de chegar ao consumidor final no tempo certo, com o mínimo custo para a empresa e com a máxima qualidade possível. A otimização de rotas é uma das áreas de maior complexidade por haver diversas variáveis que não são mensuráveis nem podem ser controladas, dependendo sempre de cada cenário. Este projeto foi desenvolvido para estudar como a empresa consegue atingir a máxima qualidade de serviço enquanto existem fatores externos e internos que influenciam diretamente a distribuição dos pedidos dos clientes, além de restrições associadas a cada cenário. Assim sendo, o principal objetivo é otimizar várias rotas de distribuição que fornecem diariamente os diversos clientes da empresa. Este estudo está inserido nos Problemas de Rotas (VRP) e começou, primeiramente, por analisar o negócio da empresa e recolher os diversos tipos de dados que posteriormente foram tratados e analisados através do Microsoft Excel e das suas funcionalidades como, por exemplo, fórmulas e tabelas dinâmicas. Por fim, foram analisadas e apresentadas soluções e cenários através de um programa específico do Microsoft Excel (VRP Spreadsheet).

This thesis is dedicated to the training and education of employees in the logistic company Gefco. The employee training in Gefco takes currently place only occasionally and unsystematically and this thesis puts forward a concept of a training system how it could look like and work in the future 6 with all different actions which are needed to be taken for the system to work properly. This theme is elaborated on the basis of technical literature in the area of theory and on the basis of own experience in the area of praxis. Training in Gefco is divided into four basic areas - general training and development, management training, language training and health and safety training together with related courses; out of these four the most voluminous part of the thesis is dedicated to the area of general training and development. The training system itself is divided into following stages - identification of training needs, scheduling of the training, realization of the training and training evaluation. Within each training area all of these stages are analyzed and it is put forward which procedures should be put into practice and what is needed to be changed and improved in order for the training in Gefco to become an effectively working system. The thesis is closed by a chapter dealing with training...

Relatório de Estágio do Mestrado em Estudos Artísticos apresentado à Faculdade de Letras
Produção e Programação nas XVII Jornadas de Cultura Popular do GEFACEste relatório é o resultado descritivo e reflexivo do meu estágio curricular de Mestrado, realizado no âmbito do 2º ciclo de Estudos Artísticos, curso pertencente ao Departamento de História, Estudos Europeus, Arqueologia e Artes, da Faculdade de Letras da Universidade de Coimbra.A entidade de acolhimento escolhida foi o GEFAC – Grupo de Etnografia e Folclore da Academia de Coimbra, assumindo eu funções na organização e programação das XVII Jornadas de Cultura Popular, evento bienal, este ano com a temática “Ofícios, Cantos e Contos: A Mulher e a Cultura Popular”. O estágio aconteceu entre 1 de dezembro de 2018 e 31 de março de 2019.Neste documento abordo a história do GEFAC, o seu trabalho etnográfico, formativo e criativo, tentando enquadrá-los historicamente e apresentando também o Estado da Arte: investigações e abordagens relativas à cultura e música tradicional portuguesa. Abordo de seguida as Jornadas de Cultura Popular, uma atividade central do GEFAC, onde procuro fazer a sua contextualização histórica e social. Finalmente para a edição deste ano, cuja organização estive envolvida, procurarei expor criticamente os processos de criação do novo espetáculo geral, a produção e programação dos restantes concertos e palestras, a divulgação e finalmente as Jornadas em si, que aconteceram de 8 de março a 5 de abril em diversos espaços culturais da cidade de Coimbra. Concluo com uma resenha descritiva do meu estágio, com todas as atividades realizadas e também um balanço final.
Prodution and Programming in Gefac’s XVII Jornadas de Cultura Popular (Folk Culture’s Journeys)This report is the descriptive and reflexive output of my Masters curricular internship, carried out under the 2nd cycle of Arts Studies, course integrated in the History, European Studies, Archeology and Arts Department of Faculty of Arts and Humanities, University of Coimbra.The chosen host entity was GEFAC - Grupo de Etnografia e Folclore da Academia de Coimbra (Ethnography and Folklore Group of Coimbra’s Academy), assuming myself roles in the organization and programming of the XVII Jornadas de Cultura Popular, biennial event, this year under the theme “Ofícios, Cantos e Contos: A Mulher e a Cultura Popular” (Crafts, Songs and Tales: Woman and the Folk Culture). The internship took place between 2018 December 1st and 2019 March 31.In this document I approach GEFAC’s history, their ethnographic, formative and creative work, framing it historically and also offering a state of art about research and approaches to the Portuguese folk culture and music. Next I approach the Jornadas de Cultura Popular, a GEFAC central activity, wich I aim to contextualize historically and socially. Finally, for this year’s edition, in which organization I was involved, I will try to expose critically the creation processes of a new general show, the production and programming of the remaining concerts and talks, the promotion and finally the Jornadas itself, that happened between March 8th and April 5th, in different cultural venues in the city of Coimbra. I conclude with a descriptive review of my internship, with all the performed activities and also a final balance.

Os resultados obtidos pela indústria automóvel revelam-se cada vez mais significativos, indicando o aumento da produção em Portugal e a adaptação dos seus meios logísticos face às suas próprias necessidades. Este comportamento demonstra que existe uma preocupação em recorrer a fornecedores que melhor se adaptem às exigências do sector automóvel. Neste sentido, foi feita uma análise da utilização do modo de transporte ferroviário de mercadorias, sendo que cada vez mais se demonstra como uma aposta compatível com um sistema de transporte sustentável ao ser conjugado com outros modos de transporte. O transporte intermodal é cada vez mais utilizado nos fluxos de distribuição, o que permite alocar cada modo de transporte a uma parte de fluxo, de acordo com as suas características. Possibilita assim aos fornecedores e clientes usufruir das vantagens que cada modo de transporte apresenta. Segundo as notícias mais recentes sobre a utilização do transporte ferroviário no âmbito de distribuição de viaturas, esta informação revela que este modo tem sido utilizado como alternativa ao transporte rodoviário em Portugal. Na sequência da greve de estivadores no porto de Setúbal, os produtores que utilizam este local como método de expedição, sentiram necessidade de recorrer a alternativas para a distribuição das suas viaturas. Veja-se o exemplo da Autoeuropa que utilizou o porto de Santander, Espanha para as suas exportações, sendo que o modo de transporte desta produção desde a fábrica até ao porto foi feito através do modo ferroviário. Esta medida alternativa serviu para identificar a praticabilidade da reactivação do transporte ferroviário entre a fábrica de Palmela e o porto de Setúbal, onde as suas conclusões permitiram no mês de Setembro de 2019 anunciar a substituição do transporte rodoviário pelo ferroviário entre o fluxo de distribuição indicado. As próprias infra-estruturas ferroviárias revelam que têm sido feitas novas alterações, criando assim futuras oportunidades para a sua utilização. Os dados do sector de distribuição em Portugal revelam que os volumes de transporte têm vindo a aumentar cada vez mais de forma anual. Este indicador permite desta forma o desenvolvimento das actividades logísticas do sector automóvel, sendo necessário garantir o funcionamento regular em conjunto com necessidades e requisitos dos fornecedores e clientes. Combinando as tendências da utilização do transporte intermodal com as oportunidades de crescimento do sector de distribuição automóvel, torna-se fundamental analisar a sua conjugação.
The results obtained by the automotive industry are becoming increasingly significant, indicating the increase of production in Portugal and the adaptation of logistics sector to its own needs. This behavior shows that there is a concern to work with suppliers that can adapt to the requirements of the automotive sector. Since the rail freight mode has become more compatible with a sustainable transport system, it was made an analysis of its use when combined with other modes of transport. Intermodal transport is increasingly used in distribution streams, which allows each mode of transport to be allocated to a flow part according to its characteristics. This enables suppliers and customers to avail the advantages that each mode of transport has. According to the latest news about the use of railroad transport in the distribution of vehicles, this information reveals that this mode has been used as an alternative to road transport in Portugal. Following the dockers strike in the port of Setúbal, producers using this location as a shipping method felt the need to resort to alternatives for the distribution of their vehicles. Take the example of Autoeuropa that appealed to the port of Santander in Spain for its exports, where the rail transport was used to carry the finished vehicles from the factory to the port. This alternative served to identify the feasibility of reactivating rail transport between the Palmela plant and the port of Setúbal, where its conclusions allowed in September 2019 to announce the replacement of road transport by rail between this distribution flow. The railway infrastructure itself shows that new changes have been made, thus creating future opportunities for its use. The data from the distribution sector in Portugal show that transport volumes have been increasing steadily on an annual basis. This indicator enables the development of logistics activities in the automotive sector, where it is necessary to ensure regular operation in conjunction with the needs and requirements of suppliers and customers. Combining intermodal transport utilization trends with growth opportunities in the automobile distribution sector, it is essential to analyze their conjugation.

Dock1 (aussi nommé Dock180) est le membre prototypique de la famille Dock d’activateurs des petites GTPases de la famille Rho. Dock1 agit au sein d’une voie de signalisation conservée au cours de l’évolution et des études génétiques ont démontré que les orthologues de Dock1, myoblast city (mbc) chez la drosophile et Ced-5 chez le nématode, activent Rac dans divers processus biologiques. Notamment, mbc est un important régulateur de la fusion des myoblastes lors de la formation des fibres musculaires de drosophile. Mbc est aussi essentiel à la migration collective d’un groupe de cellules, appelées cellules de bordures, lors de leur migration dans la chambre de l’oeuf suite à l’activation de récepteurs à activité tyrosine kinase (RTK). La migration collective des cellules de bordures récapitule certains des événements observés lorsque des cellules tumorales envahissent le tissu environnant lors de la formation de métastases. Chez les mammifères, des études réalisées en lignées cellulaires suggèrent que Dock1 est aussi un régulateur du cytosquelette lors de la migration cellulaire. De plus, certaines études ont démontré que la voie Dock1/Rac agit en aval de RTKs lors de l’invasion de cellules de glioblastome. Néanmoins, les fonctions in vivo de Dock1 chez les mammifères demeurent méconnues et le but de cette thèse est d’identifier et de caractériser certaines de ses fonctions. Guidés par la fonction de mbc, nous démontrons dans l’objectif no 1 un rôle essentiel pour ce gène au cours du développement embryonnaire grâce à la caractérisation d’une souris Dock1 knock-out. Des défauts sévères de fusion des myoblastes sont observés en absence de l’expression de Dock1 et ils contribuent à la réduction de la masse musculaire des souris mutantes. De plus, nous avons constaté une contribution du gène Dock5, un membre de la famille Dock proche de Dock1, au développement des fibres musculaires. Dans l’objectif no 2, nous avons observé que des hauts niveaux d’expression de DOCK1 corrèlent avec un mauvais pronostic chez les patientes atteintes de cancer du sein possédant une forte expression du gène codant pour le RTK HER2. Une surexpression ou une amplification du locus codant pour le récepteur HER2 est associée à près de 20% des cas de cancer du sein. Les cancers de ces patientes développent fréquemment des métastases et sont associés à un mauvais pronostic. Des études biochimiques ont révélé que DOCK1 interagit avec le récepteur HER2 dans des cellules de cancer du sein. De plus, DOCK1 est essentiel à l’activation de RAC et à la migration cellulaire induite par HER2 dans ces cellules. L’utilisation d’un modèle de cancer du sein médié par HER2 chez la souris combiné avec l’inactivation du gène Dock1 dans les glandes mammaires, nous a permis d’identifier Dock1 et Rac comme de nouveaux effecteurs de la croissance tumorale et de la formation de métastases régulées par l’oncogène HER2. Nous concluons que l’utilisation de différents modèles de souris knock-out pour le gène Dock1 nous a permis d’identifier des fonctions clés de ce gène. Tout comme son orthologue mbc, Dock1 joue un rôle important lors du développement embryonnaire en régulant notamment la fusion des myoblastes. Nos études ont également contribué à démontrer un important degré de conservation des mécanismes moléculaires de fusion entre les espèces. De plus, DOCK1 agit en aval du RTK HER2 et son expression dans les cellules épithéliales de glandes mammaires contribue au développement tumoral et à la formation de métastases induits par cet oncogène.
Dock1 (also known as Dock180) is the prototypical member of the Dock family of Rho GTPase activators (RhoGEFs). Genetic studies in Drosophila and C. elegans have demonstrated that Dock1 orthologues act upstream of the Rac GTPase to activate it during various biological processes. Myoblast city (mbc), Dock1 ortholog in the Drosophila, is an important regulator of myoblast fusion during muscle fiber formation. Moreover, mbc regulates the collective migration of a cluster of border cells downstream of the activation of some tyrosine kinase receptors (RTKs). Migration of border cells is often view as a model for studying the invasive migration of cancer cells during metastasis development. Work done in cell lines also suggests that Dock1 is an important cytoskeletal regulator that controls cell migration. The Dock1/Rac pathway was also shown to act downstream of some RTKs to promote the invasion of glioblastoma cells. Yet, the in vivo functions of Dock1 in mammals are still poorly understood and the identification and characterization of some of these functions is the main objective of my thesis. Guided by the function of mbc, in Aim #1 we revealed that Dock1 is essential to embryonic development by characterizing a Dock1 knock-out mouse model. A deficiency in myoblast fusion was observed in Dock1-null embryos which led to a reduction in their muscle mass. Furthermore, we uncovered a contribution of the other Dock1-related GEF, Dock5, to myofiber development. In Aim #2 a correlation between high level of DOCK1 expression and a poor prognosis in HER2+ breast cancer patients was revealed. Amplification or overexpression of the HER2 receptor tyrosine kinase is associated with near 20% of breast cancer cases. The presence of this genetic abnormality correlates with a poor prognosis and the development of metastasis. Biochemical and in vitro studies led us to identify that DOCK1 interacts with HER2 and is essential to HER2-mediated RAC activation and migration. The use of a HER2 breast cancer mouse model with Dock1 inactivation in the mammary gland led us to identify DOCK1-RAC signaling as novel effectors in HER2-mediated tumor growth and metastasis. We conclude that the use of Dock1 mouse models allowed us to identify some of the key functions regulated by this gene in vivo. Much like its ortholog mbc, Dock1 is essential to embryonic development and regulates myoblast fusion. Our study also reveals important degree of conservation of the mechanisms that regulate fusion between species. In addition, DOCK1 acts downstream of the HER2 RTK in mammary epithelial cells where it contributes to the progression of breast cancer pathology and the formation of metastasis induced by this oncogene.

In my theses I focus on analysis of three international private market companies. Company GEFCO ČESKÁ REPUBLIKA s.r.o. , Dalkia a.s. a ABB a.s. With this companies I'm interested in the way how they practice corporate social responsibility (CSR). When I was choosing this companies I considered facts as: Company has to have head quarter in Western Europe Company has to have experience with corporate social responsibility and has to practice CSR and has to organize social responsible activities. All chosen companies are leaders in its business sector. In my theses I will focus on overview about CSR activities of companies, mainly on corporate philanthropy. I will focus on cooperation of these companies and non government organization, I'm interested in concrete activities done by NGOs in cooperation with through CSR and how NGOs perceived activities that are done through CSR. I'm interested in the way how companies involve their employees into CSR. I 'm going to aim if subsidiaries and head quarters practice same CSR activities and using same CSR tools. I'm also interested in the role of CSR in marketing strategy of these companies. Key words Non government organization, corporate social responsibility (CSR), corporate philanthropy, corporate volunteering, volunteer, civil society, organization,...

La rapida induzione dell’affinità integrinica è un processo dinamico cruciale nel reclutamento leucocitario, che è controllato da complessi meccanismi molecolari di segnale intracellulare indotti da chemochine. Le small GTPasi della famiglia di rho e rap sono certamente le molecole di segnale più studiate in questo contesto; dati recenti da noi ottenuti hanno evidenziato inoltre un ruolo importante delle proteine tirosin-chinasi della famiglia delle Jak (Jak PTKs) che agiscono da regolatori a monte delle small GTPasi. Gli scambiatori di nucleotidi guanosinici (GEFs-Guanosine Exchange Factors) sono i principali attivatori diretti delle small GTPasi e quindi rappresentano le molecole canditate più probabili per chiarire il legame funzionale tra Jak PTKs e il modulo della rho. In questo studio abbiamo dimostrato il ruolo regolatorio concorrente di quattro differenti rho-GEFs Vav1, Sos1, Arhgef1 e Dock2 nella modulazione dell’affinità dell’integrina LFA-1 e nella conseguente adesione cellulare di linfociti T umani primari stimolati con la chemochina CXCL12. La ridotta espressione di queste quattro molecole porta ad una minore induzione dell’affinità dell’integrina LFA-1 e ad una ridotta adesione all’ICAM-1 in condizioni statiche e sotto flusso. Da notare, l’attivazione di queste quattro proteine, indotta da CXCL12, è mediata dalle Jak PTKs e avviene in un intervallo di tempo coerente con la rapida induzione dell’affinità integrinica da chemochine. Inoltre l’attivazione di RhoA e Rac1 è strettamente dipendente dall’attività di Vav1, Sos1, Arhgef1 e Dock2. Complessivamente in questo studio abbiamo identificato e caratterizzato dettagliatamente il ruolo regolatorio di quattro rho-GEFs nell’adesione mediata da LFA-1 indotta da CXCL12, fornendo una descrizione completa dei meccanismi molecolari di segnale esistenti tra Jaks e modulo della rho. Analizzando i nostri dati da un punto di vista quantitativo, abbiamo riscontrato alcune differenze tra queste proteine osservando un ruolo più marcato per Vav1 e Sos1 in confronto a quello di Arhgef1 e Dock2. Questo diverso coinvolgimento di molteplici rho-GEFs con apparentemente la stessa funzione può avvalorare la nuova interpretazione quantitativa dei meccanismi di trasduzione del segnale, dove la complessità a livello molecolare è essenziale per generare un sistema flessibile in grado di rispondere efficientemente a differenti condizioni ambientali.
The rapid integrin affinity up-regulation is a crucial dynamic process in leukocyte recruitment that is controlled by a complex inside-out signalling pathway induced by chemokines. Small GTP binding proteins of rap and rho family are certainly the most studied signaling molecules involve in this pathway; in addition our recent data identified Jak PTKs as new upstream regulator of these small GTPases. Considering that Guanosine Exchange Factors (GEFs) are the main direct activators of small GTPases, they represent obvious molecule candidates to fill out the functional gap between Jak PTKs and rho-module. In this study we show the concurrent regulatory role of four rho specific GEFs Vav1, Sos1, Arhgef1 and Dock2 in CXCL12-induced LFA-1 affinity triggering and mediated-adhesion in human T lymphocytes. A reduced expression of these four molecules resulted in an impaired chemokine-induced LFA-1 affinity up-regulation and in a reduced cell adhesion to ICAM-1 in static and under-flow conditions. Importantly, CXCL12-activation of these four proteins is mediated by Jak PTKs and occurs in a time frame coherent with LFA-1 affinity triggering by chemokine. Moreover the activation of RhoA and Rac1 is strictly dependent on Vav1, Sos1, Arhgef1 and Dock2 activity. Collectively in this study we identified and fully characterized the role of four rho-GEFs in CXCL12-induced LFA-1 mediated adhesion providing a comprehensive signalling link between Jak PTKs and rho-module. Considering our results from a quantitative point of view, we observed some variability in the relative regulatory role of these proteins, with a major role for Vav1 and Sos1 with respect to Arhgef1 and Dock2 activity. This variable involvement of multiple rho-GEFs with apparently the same function may support the new emergent quantitative-concurrency view of signal transduction in which this complexity in mechanisms controlling integrin activation is essential to generate a very flexible signalling system able to efficiently respond to a variety of environmental conditions.

The ADP-ribosylation factor (ARF) family of small G-proteins regulates membrane dynamics and intracellular membrane traffic. ARFs are activated upon GTP-binding catalyzed by guanine-nucleotide exchange factors (GEF), which works as a molecular switch and triggers association with specific target membranes. This work focused on the cloning, expression and characterization of genes from the milk yeast K. lactis and the fly D. melanogaster, which are putative homologs of the S. cerevisiae genes GEA1, GEA2, ARF1, ARF2, and ARF3.

Functional interaction of proteins is a broad term encompassing many different types of associations that are observed amongst proteins. It includes direct non-covalent interactions where the interacting proteins physically associate using an interface. There are also many protein-protein interactions where the proteins concerned are not involved in direct physical interactions but affect each other’s functions. Central focus of this thesis is to understand the various aspects of functionally interacting proteins. Chapter 1 of this thesis provides an introduction to functional interactions between proteins and discusses the key developments available in the literature. This chapter discusses the different types of functional associations observed commonly between proteins. Various approaches developed over time to elucidate such interactions have also been discussed. This chapter highlights how functional interactions between proteins have been helpful in understanding different cellular processes such as organization of metabolic pathways. The chapter emphasizes the importance of functional interactions between proteins, providing a motivation for development of methods with enhanced accuracy and sensitivity for the prediction of functional interactions. In this thesis, domain families which are found to co-exist in multidomain proteins have been used to understand and subsequently predict functional associations amongst proteins. Domains in proteins typically serve as modules associated with specific functions. There exist proteins with a single domain which describes the entire function of a protein, while there also exist proteins containing multiple domains, where various domains in unison describe the complete function of the multidomain protein. Therefore, by virtue of “guilt by association” domain families found together in multidomain proteins are functionally linked. This forms the basic premise for understanding functional association amongst proteins and is explained in great detail in the Introduction chapter. Using domain families which co-occur in multidomain proteins as the basis for functional association has many merits. First, as stated before, constituent domain families act as effective descriptors of function(s) of proteins. For example, members of SH3 domain family mediate protein-protein interactions by binding to regions with polyproline conformation irrespective of the multidomain protein in which it occurs. Thus, studies of domain families co-existing in multidomain proteins act as an accurate resource of functional associations between proteins. Also, assignment of domains to a protein relies on homology detection which has achieved a high level of reliability, thus, resulting in reasonably accurate prediction of functions. Such approaches enable exhaustive coverage of many diverse proteins including many multidomain proteins leading to detection of large numbers of functional associations between domains of multidomain proteins. Given the advantages attributed to functionally linked domain families in further understanding of functional associations, it is imperative to exhaustively enumerate all possible pairs of functionally linked domain families in multidomain proteins and study their various properties. This aspect is covered in the second chapter of the thesis. In the second chapter, analysis of domain families which co-occur in multidomain proteins, termed as 'tethered domain families', has been reported. For this analysis, a large dataset of multidomain proteins was considered from a diverse set of fully sequenced genomes from many eukaryotic and prokaryotic organisms. In every multidomain protein, all possible pairs of unique domain family pairs have been considered and they are assumed to be under the same functional/evolutionary constraint. Thus, from the entire dataset of multidomain proteins, all possible pairs of tethered domain families are obtained. For a given domain family, the number of other uniquely tethered families is referred to as the tethering number of a domain family. Therefore, tethering number of a domain family is an indicator of the diverse functional contexts in which a particular domain family is involved. Further analysis was carried out to understand various other attributes of domain families and its relation to tethering number. The results are summarized in the following points: 1) Distribution of tethering numbers of domain families in the entire dataset is found to be highly heterogeneous. Nearly 88% of domain families (10783 out of 12249 domain families) have tethering number of 10 or less and only 78 domain families show more than 100 unique associations. Further analysis reveals bias in functions of families showing high and low tethering numbers. The domain families with high tethering numbers are involved in processes such as signaling and protein-protein interactions. The domain families with low tethering numbers are often found to be involved in metabolic processes. 2) Differences are also observed in the type of organisms containing the domain families and their tethering numbers. Typically, domain families with high tethering numbers are ubiquitously found across almost all the kingdoms of life. In contrast, most of the domain families exclusively found in a kingdom have low tethering numbers. Furthermore, for the ubiquitously occurring domain families with high tethering numbers, the number of associations made and the type of associations are not strictly conserved across the kingdoms. Thus, the tethering preferences of such domain families vary across the kingdoms depending on their function. For instance, the protein kinase domain family which is a key regulator of signaling processes in eukaryotes, has a high tethering number in eukaryotes (270), and low tethering number in prokaryotes (96). 3) Tethering number of domain families is found to be correlated with the number of members (population) comprising a family. A Pearson correlation coefficient of 0.78 at a p-value ≤0.001 is obtained for the correlation between tethering number of domain families and their population. 4) Tethering numbers of domain families are also found to be well correlated with sequence and functional diversity within families. Thus, domain families with high tethering numbers comprise of members showing diversity in both sequence and functions. Thus, the work presented in second chapter provides a framework for understanding the tethering preferences of domain families. The use of tethered domain families to identify functional association amongst proteins is the central theme of third and fourth chapters of this thesis. The use of tethered domain families for the prediction of functionally interacting proteins originates from the initial idea of “Rosetta stone” approach, which was proposed by Ouzounis and coworkers and Eisenberg and coworkers in 1999. Rosetta stone approach demonstrated the use of fused genes in predicting functional interaction. It stems from the observation that in many organisms, genes corresponding to proteins acting in a metabolic pathway are found fused in another organism. Thus, enumeration of 'fused genes' in a template database could provide a good basis for prediction of functionally interacting proteins in target organisms in which the homologous genes are not found to be fused. The method has been shown, by others, to work quite effectively in prokaryotes, especially in the identification of interactions between metabolic proteins. Chapter 3 of this thesis explores the idea of “Rosetta stones” at the level of domain families, by considering tethered domain families as analogs to the fused genes. In this analysis, tethered domain families derived from multidomain proteins comprises the template dataset. If members of two domain families occurring in a multidomain protein are found to occur independently in two different proteins in the target organism then an interaction is predicted between these two proteins (collection of such predicted interactions is henceforth referred as TEDIP database, Tethered Domain-based Interaction Prediction). During this analysis, care is taken such that none of the proteins in the template dataset belongs to the target organisms. The entire analysis has been conducted on 6 model organisms which act as the target dataset where functional interactions between proteins are predicted. The effectiveness of tethered domain families in functional interaction prediction is compared with two other datasets 1) all experimentally known interactions and 2) interactions predicted on the basis of their homology with interacting domain families with known structure. Subsequently, an attempt has been made to answer these questions: 1) how effective is the information on tethered domain families in predicting functional linkages amongst proteins operating in pathways in eukaryotic organisms? 2) what is the false positive rate of the predictions? The above mentioned datasets show very little overlap in the coverage of functional interactions. This is largely attributed to insufficient sampling and inherent bias existing in each of the methods. The TEDIP datasets in the six organisms led to an average three-fold more functional interaction predictions in cellular pathways than the other two datasets. Nearly 90% of the predicted interactions derived from tethered domain families are amongst proteins across different pathways. In yeast, more than 60% of such interactions were found to be overlapping with a recent large scale genetic interaction screen based on synthetic lethality especially performed for metabolic proteins, thus establishing the effectiveness of this approach in understanding pathway crosstalk. Along with efficacy in identifying functional interactions, an assessment based on co-localization, co-expression and overall functional similarity based on Gene Ontology (GO) terms was carried out. It was found that the TEDIP predictions and experimentally found interactions show poor correspondence with co-expression and co-localization data (10% and 20% respectively for the two methods). Additionally, it was found that functional similarity between predicted interacting proteins in TEDIP dataset is low (5%) and is comparable to experimentally known interactions that shows 10% similarity in functions based on a scoring function for GO term similarity. From Chapter 3, it was concluded that the use of tethered domain families is effective in exhaustive enumeration of functionally associated proteins. However, the low co-expression and functional similarity measures are a cause for concern. On the one hand, co-expression and GO functional similarity have been found to be weak predictors of functional interactions, explaining the low values obtained for both predictions in the TEDIP datasets and experimentally known interactions. On the other hand, the poorer values shown for predictions in the TEDIP datasets suggest that further improvement in prediction accuracy is possible. Chapter 4 explores the use of machine learning in improving the accuracy of functional interaction prediction based on TEDIP dataset. In Chapter 4, two distinct machine learning approaches have been employed on a training dataset derived exclusively from yeast. Since the objective of the work is to improve the accuracy of prediction of functional interactions, the GO based functional similarity measures have been used to define positive and negative datasets. Thus, in the training dataset, positive interactions comprises of protein pairs which show high GO similarity in functions as defined in chapter 3 and 10% of this data overlaps with experimentally known interactions, while the negative dataset consists of protein pairs with no or insignificant similarity in their functions and additionally do not show similarity to any experimentally known interactions. Two machine learning approaches, namely Support vector machine (SVM) and Random forest, have been used on this training dataset. Use of two distinct approaches helps in addressing the weakness, if any, of these methods. Fourteen carefully chosen features have been utilized during the training process to aid in the process of distinguishing potentially correctly predicted interactions from incorrect predictions. Out of 14 features, some of the features chosen for the analysis are involved in quantifying the extent of similarity between the template proteins containing the fused domain families and the target protein pairs predicted to interact. The analysis also incorporates graph theory based parameters which are derived from a domain family based graph. In such a graph, each of the domain families which are involved in forming multidomain proteins represents the nodes and an edge is constructed between domain families which are found to co-exist in at least one multidomain protein. Graph theory based parameters such as clustering coefficient, degree and topological overlap have been employed. These are useful in down weighting appropriately the domain family pairs showing large number of associations which are expected to be promiscuous in their functions. These features also enable in identifying domain family pairs which are functionally related. Apart from the above mentioned features, coevolution and phylogenetic profiling of tethered domain families is also utilized to identify functionally related domain family pairs. Utilizing all these features in training, the machine learning approach yielded an accuracy of 94% using SVM and 92% using Random forest against the training data. Furthermore, the importance of using all these features has been addressed by performing principle component analysis, training both SVM and Random forest by removing one feature at a time and by quantifying the sensitivity by using only one feature. All of these suggest that the features used provide non-redundant information and contributed significantly to the classification. The models so generated were finally used on all the predicted functional interactions after the removal of the training dataset in yeast. The true positives observed were 56% using SVM and 63% using Random forest with around 80% of the interactions common between the two methods. Further analysis has been carried out on these interactions by first imparting a confidence score to these interactions using support vector regression that provides a probabilistic measure for SVM classification. Based on a cutoff of 0.5, 62455 interactions in total were termed as high confidence interactions. Further analysis was carried out for the high confidence interactions. Out of these, in 2855 interactions, both the proteins predicted to interact could be associated with a pathway in KEGG database. In-depth case studies have been performed on this dataset of 2855 interactions. Literature mining suggested that many known cross-pathway interactions such as between TCA and glycolysis are captured as high confidence interactions using TEDIP dataset. A few other case studies of high confidence interactions with supporting literature evidence are also presented in the chapter. These predictions could further aid in experimental characterization of pathway cross-talk between important metabolic and signaling pathways. So far, the thesis discussed analyses involving functional interactions and their prediction. In the subsequent chapters, analyses pertaining to two different types of functional interactions are discussed. Chapters 5 and 6 involve analyses incorporating metabolic proteins in diverse pathways in the pathogenic organism Plasmodium falciparum. Chapter 5 attempts to improve the coverage of the repertoire of metabolic proteins in P.falciparum while in Chapter 6 interactions and pathways prevalent in different stages in the life cycle of the parasite are deciphered and discussed. Apart from functionally interacting proteins in metabolic pathways, physically and transiently interacting proteins have been analyzed and discussed in Chapters 7 and 8. In Chapter 5, metabolic proteins participating in pathways in Plasmodium falciparum have been analyzed. P.falciparum is the causative agent of malaria, a disease which affects large populations in the subtropical regions. P.falciparum genome is atypical and is rich in Adenine/Thymine pairs, and there is presence of large stretches of amino acid repeats encoded in protein coding regions. Various sequence-related features of P.falciparum proteins when compared with those of other organisms show extensive divergence. All of these have made reliable function prediction, by homology to other proteins with known functions, daunting. Like other proteins in P.falciparum, metabolic proteins have also diverged significantly from their functional counterparts in model eukaryotes such as yeast. Metabolic pathways play an important role in the survival of the organism and hence are amenable towards the identification of proteins susceptible to drugs, thereby combating pathogenesis. Chapter 5 of the thesis aims at furthering knowledge pertaining to metabolic proteins by first quantifying the extent of divergence observed in the already characterized metabolic proteins. This knowledge is further used in identification of potential metabolic proteins which are not identified as proteins involved in metabolic pathways by other annotation efforts undertaken for P.falciparum. In the first part of the chapter, the extent of divergence in the sequences of metabolic proteins in P.falciparum has been determined by comparing the P.falciparum proteins with their functional counterparts from 34 completely sequenced unicellular eukaryotic organisms. Comparison of domain architectures between the P.falciparum proteins with their functional counterparts reveals that in nearly 54% of metabolic pathways, proteins show nearly the same domain architecture as the other functional counterparts. Inversion, deletion and duplication of domains are observed in rest of the proteins. Further analysis reveals that P.falciparum proteins are longer than their functional counterparts. It was also observed in nearly 15% of the cases, the domains are characterized by the presence of large non-conserved or plasmodium genus specific inserts within the domain assigned regions. There is also prevalence of unassigned regions in the N- and C- terminal regions in P.falciparum proteins when compared with their functional counterparts. Finally, it was also observed that metabolic proteins of P.falciparum show significantly low sequence similarity when compared with other functional counterparts. From this analysis, it can be clearly seen that metabolic proteins of P.falciparum have significantly diverged from such proteins in other organisms, thus making function prediction by homology very difficult. There are several steps in metabolic pathways in P.falciparum which are expected to be active based on experimental analysis. However, some of these proteins with expected functions have not been identified so far. One of the reasons for this apparent incompleteness is the high divergence observed in the metabolic proteins of P. falciparum. To overcome this limitation, in the second part of the chapter, a sensitive approach based on domain family assignment (MulPSSM), developed in-house, has been used to identify proteins which are potentially involved in metabolic pathways. The approach is based on reverse PSI–BLAST, where multiple sequence profiles for each family are used to search against sequence databases. This approach has been shown to be better or at-par with other remote homology detection procedures. Using this approach, 15 P. falciparum proteins have been identified which can potentially function as metabolic proteins and were not characterized in P.falciparum so far. All the proteins identified by the approach show low sequence similarity to other well characterized proteins and contain significant fractions of unassigned regions thus, making function recognition non-trivial. Supporting literature and other data is provided to demonstrate the robustness of the homology-based annotation of the identified pathway proteins. Chapter 6 is an analysis of the dynamic changes occurring in the metabolic network of P.falciparum during its life cycle. In this chapter, two aspects of P. falciparum metabolic proteins have been integrated and analyzed. First, the dataset of protein-protein interactions derived from experimental studies and second, the datasets of microarray analysis providing information on stage specific expression of P. falciparum genes corresponding to the metabolic proteins. As a first step, protein-protein interaction information for the metabolic proteins was gathered. A total of 810 interactions have been obtained, where one or both proteins are involved in a pathway. Subsequently, these interactions were compared with 14070 interactions involving metabolic proteins from free-living and non-pathogenic unicellular eukaryote yeast. Comparison across the two organisms shows wide discrepancy in the number of proteins involved in interactions and also the pathways in which they participate. Out of the 810 interactions in P.falciparum, 173 are found uniquely in plasmodium where both or one of the protein have no identifiable homolog in yeast. Insufficient sampling of interactions made by proteins in P.falciparum in comparison to yeast, is one of the reasons for the observed discrepancy. However, the differences due to the parasitic lifestyle of P.falciparum could also be a potential reason. Further analysis of the protein-protein interactions by the metabolic proteins revealed that a large fraction of interactions are made between a metabolic protein and a non-metabolic protein. For instance, interaction observed between glycolytic protein phospoglycerate kinase with MAP kinase. This trend is observed in both plasmodium and yeast where 65% and 77% of the interactions, respectively, involve proteins not directly participating in metabolic pathways. Further, interactions between proteins belonging to different pathways and lastly, interactions between proteins in the same pathway are uncovered. All of these interactions depict the different modes by which metabolic pathways are regulated through protein-protein interactions. Another aspect explored in this analysis is the stage specific expression of genes encoding these metabolic proteins. The analysis is especially relevant in the parasite because its entire life cycle is divided into seven distinct stages. Upon integrating the protein-protein interactions with the gene expression data, it became apparent that the trophozoite, schizont and gametocyte stages show large fractions of co-expressed genes encoding proteins involved in protein-protein interactions within metabolic pathways. The high preponderance of co-expressed genes encoding for interacting protein pairs in these stages is also consistent with metabolic requirement of plasmodium in the various stages. Glycolytic pathway is central to energy production in the parasite and is discussed at length in this chapter. Members of this pathway are involved in interactions with other glycolytic proteins (9 such interactions), they also interact with proteins involved in other pathways (30 interactions) and with proteins not involved directly in any metabolic pathway (75 interactions). Nearly 70% of the interactions made by the glycolytic proteins are encoded by genes found to be co-expressed across the various stages. Integration of gene expression data along with protein-protein interaction information for metabolic pathways such as the glycolytic pathway thus, highlights the complex mode of regulation underlying this pathway. The analysis carried out in this chapter emphasizes on the intricacies involved in the regulation of metabolic proteins in P.falciparum. Chapter 7 describes an in-depth analysis carried out to understand the basis for interaction specificity between small monomeric GTPases and their regulators, the Guanine nucleotide Exchange Factors (GEFs). Monomeric GTPases are involved in binding to guanine nucleotide. These proteins can bind to both GTP and GDP. However, transition from GDP bound to GTP bound form occurs with large conformational changes and requires binding of the GEFs. The conformational changes that arise due to the nucleotide exchange are required for the GTPases to bind to its various effectors. For the analysis carried out in Chapter 7, GTPases belonging to the Ras superfamily have been considered. The superfamily is further subdivided into 5 distinct families based on their functions. The 5 families are Ras, Ran, Rab, Arf and Rho. Members belonging to each of these families are involved in a wide array of cellular processes such as signaling and cytoskeletal remodeling. Members of each of these GTPase families bind to structurally distinct GEFs, and in some cases, multiple GEFs are involved in nucleotide exchange within a family. It is intriguing therefore, to understand how GTPases belonging to the same structural family maintain specificity across the highly dissimilar GEFs and this forms the main objective of this analysis. So far, 13 distinct complexes between GTPases and their cognate GEFs have been solved using X-ray crystallography. This set of structural complexes forms the starting point of the analysis. As a first step, pairwise structural comparison of the interfaces has made between various pairs of complex structures. Based on these comparisons, it is apparent that most of the interfaces in the GTPase and GEF complexes comprise of residue positions which are topologically not equivalent suggesting different modes of binding across these complexes. Further analysis was carried out to probe the extent of specificity underlying these complexes. This is achieved by determining interface residues which are found to be conserved in a family specific manner. Such residue positions have been obtained by using a statistically robust algorithm Contrast Hierarchical Alignment and Interaction Network (CHAIN) that extracts sequence patterns most distinguishing two sets of homologous sequences. The analysis indicated the presence of family specific residues at the GTPase and GEF interface. Such residues could be implicated in maintaining the specific interactions between the GTPases and the GEFs. The robustness in the specificity of the interactions was further interrogated by providing an energetic basis to the specificity in the interactions mediated by the cognate GTPases and the GEFs and also understanding how crosstalk is prevented across the non-cognate complexes. For each of the 13 cognate complexes, empirical interaction energies have been estimated using FoldX. The interaction energy is compared to non-cognate complexes which are obtained by swapping the interface residues of the cognate GTPase with the non-cognate GTPase residues. For most of the complexes, it was observed that the interaction energies for the cognate complexes are much lower than the non-cognate complexes. Energy values across the non-cognate complexes are usually indicative of reduced stability, thereby precluding such interactions from occurring. Such large energy differences between cognate and non-cognate interactions arise due to drastic substitutions at the interface patch due to difference in the charge or other stereochemical aspects of the amino acids. Both evolutionary and energy based analysis indicates the presence and importance of few family specific residues in the cognate complexes and also the presence of unfavorable residues in the non-cognate complexes thus preventing crosstalk. However, apart from changes at the interfaces, many positions outside the interface also undergo changes across the various homologs within the same family/subfamily of GTPase. Coevolutionary analysis of GTPase and GEFs from multiple eukaryotic organisms has been carried out in these complexes and it was observed that most of the coevolving positions are not found at the interface. Many of these residue positions are near the active site or near the interface. Identification of such coevolving positions, where residue variations in the GTPase are strongly coupled to the GEF, may provide initial clues to the possible allosteric path adopted in connecting the binding of GEF to the vast structural changes observed during GTP exchange in GTPases. Thus, the analysis provides a comprehensive framework to understand how interaction specificity has evolved between the GTPase and GEF complexes. Chapter 8 discusses another example of transient protein-protein interaction observed between proteins implicated in signaling process in Dictyostelium discoideum. The work reported in this chapter was carried out in collaboration with Prof. Nanjundaiah and coworkers from Molecular Reproduction and Developmental Genetics department, Indian Institute of Science. All the experimental analyses mentioned in this chapter were carried out by Prof. Nanjundaiah and coworkers and the author carried out all the computational analysis. Experimental analysis indicated the presence of a ribosomal protein S4 in D. discoideum which mediates interactions with CDC24 and CDC42. The protein is speculated to be a functional analog of yeast scaffolding protein Bem1. However, the exact structural and sequence features of the protein which can accommodate its non-ribosomal function as a scaffold by mediating protein-protein interactions are not clearly understood. With the aid of structural modeling, a 3-D structure was generated for the C-terminal regions of D. discoideum protein S4. The modeled structure, as in the template used for modelling, resembled the fold of SH3 domain which has been shown to be involved in protein-protein interactions. Structural and sequence analyses were carried out to evaluate the potential mode by which interactions could be mediated by this protein. The hypothesis generated was further corroborated by experimental analysis. Thus, both experimental and computational analysis provide evidence for the functional role of the ribosomal protein S4 from Dictyostelium discoideum as a scaffold. Chapter 9 summarizes the conclusions reached in various chapters of the thesis. The thesis embodies analyses probing various aspects of functional interactions between proteins. A frame work has been provided to elucidate functional interactions using tethered domain families in multidomain proteins. Further, the role of these functional interactions have been explored in different scenarios by exhaustively analyzing metabolic proteins and their regulation in pathogenic organism Plasmodium falciparum and by also analyzing two distinct types of transient protein-protein interactions.