A determinação dos marcadores sorológicos P-ANCA (anticorpo perinuclear contra estruturas citoplasmáticas do neutrófilo) e ASCA (anticorpo anti-Saccharomyces cerevisiae) auxilia de forma menos invasiva no diagnóstico da doença inflamatória intestinal (DII). O padrão de associação mais relacionado à retocolite ulceratica inespecífica (RCUI) ocorre com ASCA- (negativo) e P-ANCA + (positivo). Na doença de Crohn (DC) ocorre o contrário, ou seja, ASCA+ e P-ANCA-. O P-ANCA é determinado por imunofluorescência indireta usando neutrófilos fixados em etanol, e o ASCA através de ELISA. De forma geral, a prevalência do P-ANCA em pacientes com RCUI tem variado entre 50 e 80% e em pacientes com DC entre 10 e 30%. Controles sadios têm revelado prevalência menor que 4% e controles patológicos em torno de 8%. Alguns trabalhos mostraram ampla variação nos resultados, sugerindo além de variação genéticas, variações metodológicas de acordo com a população estudada. Foram realizadas análises no soro de 200 pacientes para pesquisa de P-ANCA e ASCA, sendo 98 com RCUI e 102 com DC. O grupo controle foi representado por 54 indivíduos sadios. Os pacientes com DII foram oriundos do ambulatório de Gastroenterologia ? Grupo de Intestino do Hospital das Clínicas Faculdade da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP). A prevalência encontrada do P-ANCA na RCUI, DC e grupo controle correspondeu a 61,2%, 16,7% e 5,6% respectivamente, enquanto que a do ASCA para a DC, RCUI e grupo controle correspondeu a 52,9%, 27,6% e 5,6% respectivamente. A sensibilidade e especificidade encontrada com o padrão ASCA+/P-ANCA- para DC foi de 45,1% e 89,% respectivamente, enquanto que para o padrão P-ANCA+/ASCA- para RCUI foi de 43,9% e 91,2% respectivamente. No que diz respeito às características clínicas e demográficas, nenhuma associação com a presença dos anticorpos foi estabelecida no presente estudo, exceto quando avaliado o uso de drogas imunossupressoras e infliximab em pacientes com DC e ASCA+ , a qual se mostrou aumentada (p=0,008). Assim sendo, a determinação dos anticorpos P-ANCA e ASCA na DII possui baixa sensibilidade, mas níveis elevados de especificidade como demonstrado em outros estudos publicados. A correlação dos anticorpos P-ANCA e ASCA com características clínicas ainda permanece controversa. / The determination of serologic markers P-ANCA (perinuclear antineutrophil cytoplasmic autoantibodies) and ASCA (anti-Saccharomyces cerevisiae mannan antibodies) assists as non-invasive way on the inflammatory bowel disease (IBD) diagnosis. The most associated pattern to ulcerative colitis (UC) occurs with ASCA- (negative) and P-ANCA + (positive). In the Crohns disease (CD) is the opposite, that is, ASCA+ and P-ANCA-. Determination of P-ANCA is performed by an indirect immunofluorescence assay, using ethanol-fixed neutrophil slides and ASCA is measured by ELISA. Usually, the prevalence of P-ANCA in patients with UC has varied between 50 and 80% and in patients with CD between 10 and 30%. Healthy controls have disclosed lesser prevalence than 4% and pathological controls around 8%. Some studies had shown a wide variation in the results, suggesting both genetic and methodologic variations, according to the studied population. Serum samples were obtained from 200 patients for analysis of P-ANCA and ASCA, being 98 with UC and 102 with CD. The control group was represented by 54 healthy individuals. Patients with IBD were selected from the Department of Gastroenterology - Intestine Group of the Hospital das Clínicas of the University of São Paulo (HCFMUSP). P-ANCA prevalence found in UC, CD and control group were 61,2%, 16,7% e 5,6%, respectively, but ASCA prevalence in UC, CD and control group were 52,9%, 27,6% e 5,6%, respectively. Sensitivity and specificity achieved using ASCA+/P-ANCA- pattern for CD were 45,1% e 89,%, respectively, but P-ANCA+/ASCA- pattern for UC were 43,9% e 91,2% respectively. In this study, the current data did not support a relationship between the serological markers and clinical and demographic characteristics, except when evaluated the use of immunosuppresive drugs and infliximab in patients with CD and ASCA+, which were increased (p=0,008). Thus, the determination of antibodies P-ANCA and ASCA in the IBD gets low sensitivity, but high levels of especificity as demonstrated in other published reports. The correlation of antibodies P-ANCA and ASCA with clinical characteristics appears to be limited.

Pleckstrin homology (PH) domains, which play an essential role in membrane trafficking and signal transduction, recognize phosphoinositides with a diverse range of affinities and specificities. The PH domains of the Grp1 family of Arf GTPase exchange factors recognize a select group of phosphoinositides with dramatic differences in specificity, despite 90% sequence identity. The work described in this thesis has focused on the structural basis for these differences. The structure of the Grp1 PH domain revealed structural determinants for phosphoinositide recognition. Through a wide range of crystallographic and biochemical means, the structural basis that accounts for the differential binding affinities amongst the Grp1 family PH domains has also been determined. Furthermore, examination of the structural details of these PH domains bound to different inositol phosphate groups have aided in understanding the structural mechanisms by which all PH domains recognize phosphoinositides.

Dans cette thèse, nous abordons deux problèmes de théorie des graphes liés à deux problèmes biologiques de symbiose (deux organismes vivent en symbiose s'ils ont une interaction étroite et à long terme). Le premier problème est lié au phénomène de l'Incompatibilité cytoplasmique (IC) induit par certaines bactéries parasites chez leurs hôtes. L'IC se traduit par l'impossibilité de donner naissance à une progéniture saine lorsqu'un mâle infecté s'accouple avec une femelle non infectée. En termes de graphe ce problème peut s'interpréter comme la recherche d'une couverture minimum par des "sous-graphes des chaînes" d'un graphe biparti. Un graphe des chaînes est un graphe biparti dont les noeuds peuvent être ordonnés selon leur voisinage.En terme biologique, la taille minimale représente le nombre de facteurs génétiques impliqués dans le phénomène de l'IC. Dans la première moitié de la thèse, nous abordons trois problèmes connexes à ce modèle de la théorie des graphes. Le premier est l'énumération de tous les graphes des chaînes maximaux arêtes induits d'un graphe biparti G, pour lequel nous fournissons un algorithme en delai polynomial avec un retard de O(n^2m) où n est le nombre de noeuds et m le nombre d'arêtes de G. Dans la même section, nous montrons que (n/2)! et 2^(\sqrt{m}\log m) bornent le nombre de sous-graphes de chaînes maximales de G et nous les utilisons pour établir la complexité "input-sensitive" de notre algorithme. Le deuxième problème que nous traitons est de trouver le nombre minimum de graphes des chaînes nécessaires pour couvrir tous les bords d'un graphe biparti.Pour résoudre ce problème NP-hard, en combinant notre algorithme avec la technique d'inclusion-exclusion, nous fournissons un algorithme exponentiel exact en O^*((2+c)^m), pour chaque c > 0 (par O^* on entend la notation O standard mais en omettant les facteurs polynomiaux). Le troisième problème est l'énumération de toutes les couvertures minimales par des sous-graphes des chaînes. Nous montrons qu'il est possible d'énumérer toutes les couvertures minimales de G en temps O([(M + 1) |S|] ^ [\ log ((M + 1) |S|)]) où S est le nombre de couvertures minimales de G et M le nombre maximum des sous-graphes des chaînes dans une couverture minimale. Nous présentons ensuite la relation entre le second problème et le calcul de la dimension intervallaire d'un poset biparti. Nous donnons une interprétation de nos résultats dans le contexte de la dimension d'ordre / In this thesis, we address two graph theoretical problems connected to two different biological problems both related to symbiosis (two organisms live in symbiosis if they have a close and long term interaction). The first problem is related to the size of a minimum cover by "chain subgraphs" of a bipartite graph. A chain graph is a bipartite graph whose nodes can be ordered by neighbourhood inclusion. In biological terms, the size of a minimum cover by chain subgraphs represents the number of genetic factors involved in the phenomenon of Cytoplasmic Incompatibility (CI) induced by some parasitic bacteria in their insect hosts. CI results in the impossibility to give birth to an healthy offspring when an infected male mates with an uninfected female. In the first half of the thesis we address three related problems. One is the enumeration of all the maximal edge induced chain subgraphs of a bipartite graph G, for which we provide a polynomial delay algorithm with a delay of O(n^2m) where n is the number of nodes and m the number of edges of G. Furthermore, we show that (n/2)! and 2^(\sqrt{m} \log m) bound the number of maximal chain subgraphs of G and use them to establish the input-sensitive complexity of the algorithm. The second problem we treat is finding the minimum number of chain subgraphs needed to cover all the edges of a bipartite graph. To solve this NP-hard problem, we provide an exact exponential algorithm which runs in time O^*((2+c)^m), for every c>0, by a procedure which uses our algorithm and an inclusion-exclusion technique (by O^* we denote standard big O notation but omitting polynomial factors). Notice that, since a cover by chain subgraphs is a family of subsets of edges, the existence of an algorithm whose complexity is close to 2^m is not obvious. Indeed, the basic search space would have size 2^(2^m), which corresponds to all families of subsets of edges of a graph on $m$ edges. The third problem is the enumeration of all minimal covers by chain sugbgraphs. We show that it is possible to enumerate all such minimal covers of G in time O([(M+1)|S|]^[\log((M+1)|S|)]) where S is the number of minimal covers of G and M the maximum number of chain graphs in a minimal cover. We then present the relation between the second problem and the computation of the interval order dimension of a bipartite poset. We give an interpretation of our results in the context of poset and interval poset dimension... [etc]

Indiana University-Purdue University Indianapolis (IUPUI) / Giant axonal neuropathy (GAN) is a rare genetic disease that causes progressive damage to the nervous system. Neurons in GAN patients develop an abnormal organization of cytoskeletal proteins called intermediate filaments (IFs), which normally provide strength and support for the overall cell structure. The irregular IF structure in GAN patient neurons leads to a progressive loss of motor skills in children and subsequent death in adolescence. GAN is caused by reduced levels of the gigaxonin (Giga) protein. Giga functions to control the degradation of other cellular proteins, and the loss of Giga in GAN cells results in significantly elevated levels of the galectin-1 (Gal-1) protein. Gal-1 stabilizes the active form of the Ras signaling protein, which functions as a molecular switch to regulate the phosphorylation and subsequent organization of IFs. The connection between these pathways led us to propose that Giga regulates IF phosphorylation and structure by modulating Ras signaling through the degradation of Gal-1. Using GAN patient cells, we demonstrated that restoring Giga reduced Gal-1 protein levels, decreased IF phosphorylation, and reestablished normal IF organization. Similar effects of reduced IF phosphorylation and improved IF structure were also obtained in GAN cells by directly decreasing the protein levels of either Gal-1, or downstream Ras signaling proteins. Taken together, these results demonstrate that the loss of Giga induces Gal-1 mediated activation of Ras signaling, thereby leading to the increased IF phosphorylation and abnormal IF structure observed in GAN cells. Identification of aberrant Ras signaling is significant because it is the first to specify a mechanism by which the loss of Giga leads to the development of GAN and provides targets for novel drug therapies for the treatment of this currently immedicable genetic disease.

Stomata are small pores that are located on the surface of epidermal leaves, and they can regulate the uptake of CO2 and prevent water lose by opening and closing the pores. Stomata of plants can be regulated by external condition such as CO2, biotic and abiotic stresses and internal factors. CAM (crassulacean acid metabolism) plants adapt to hot and dry environments by closing stomata during the day and opening stomata during the cool night. However, it is still unclear how CAM plants open their stomata during the night and close them during the day. In this study, a number of factors were evaluated for their potential roles in promoting stomatal opening in the model CAM plant Kalanchoe laxiflora. Citrate is an important organic acid and it accumulates during the night in CAM plants. It is shown in this study that citrate promoted stomatal opening in detached leaf epidermis of Kalanchoe laxiflora. Further, the cytokinin zeatin is also shown to stimulate stomatal opening in detached leave of Kalanchoe laxiflora. Melatonin is an important regulator of circadian rhythms in mammals and has been implicated in regulation of plant abiotic stress responses. Melatonin was detected in the leaves of Kalanchoe laxiflora. It promoted stomatal opening in detached epidermis of Kalanchoe laxiflora. Together, these results suggest that stomata of Kalanchoe laxiflora respond to citrate and malate which are the main organic acids accumulate during nighttime and also to some signaling molecules (zeatin, melatonin, and serotonin) by opening stomata during dark period.

<p dir="ltr">Plant immunity to pathogens involves a network of genetic, molecular, and cellular processes that culminate in activation of responses that restrict pathogen ingress and reduce diseases symptoms. Mechanisms of pathogen recognition, signaling, and activation of immune responses are well understood especially for (hemi) biotrophic pathogens. By contrast, there is paucity of knowledge on immune signaling for responses to broad host necrotrophic fungi such as <i>Botrytis cinerea (Botrytis</i><i>)</i>. Plant resistance to such pathogens is genetically complex with no complete resistance observed in any host species. However, genetic variation for quantitative resistance has been documented although the molecular mechanisms are poorly understood. In the current study, we focused on functional dissection of components of tomato immune signaling underlying quantitative resistance to <i>Botrytis</i>. Tomato BIK1-family receptor-like cytoplasmic kinases (RLCKs) were studied using gene edited mutants, and subsequent molecular, biochemical, and genomic characterizations of the mutants and the corresponding proteins. In addition, <i>Botrytis</i> induced transcriptome of Arabidopsis and tomato were compared to explain observed differences in their resistance to the fungus. The results from these studies are described in four chapters. Chapter 1 provides a review of RLCKs and their function in plant responses to biotic and abiotic stresses. Chapter 2 presents the genetic, molecular, and biochemical characterization of tomato RLCK, TPK1B RELATED PROTEIN KINASE (TPK09) in fungal resistance and responses to light stress. Chapter 3 highlights observations on the functions of TPK1B RELATED PROTEIN KINASE7 (TPK07) in fungal and bacterial resistance. Lastly, Chapter 4 covers comparative transcriptome analysis of Arabidopsis and tomato responses to <i>Botrytis</i>.</p><p dir="ltr">Specialized cell-surface receptors mediate the perception of environmental changes. A subset of plant cell surface receptors recognizes pathogen-associated molecular patterns (PAMPs), which are immunogenic pathogen or host-derived molecules, peptides (phytocytokines), lipids, and carbohydrates. PAMPs are perceived by surface receptors designated as pattern recognition receptors (PRRs) that are categorized as receptor like kinases (RLKs) or receptor like proteins (RLPs). Recognition of PAMPs through PRR is an evolutionarily conserved pathway that aids plants in specific recognition of pathogens. The signaling events initiated by PRRs are connected to PRR-associated RLCKs, which amplify the signal and activate other regulatory proteins. PRR-RLCK activation is linked to immune messengers ROS, Ca⁺, and MAPKs based on extensive research in Arabidopsis which also established the paradigm for RLCKs functions in integrating signals from various PRRs. However, insufficient attention was given to RLCK functions in crop plant responses to biotic and abiotic stressors and, thus, limited data exists on RLCKs from crops of agronomic and horticultural significance. To address this gap, we conducted genetic, genomic, and molecular studies on the biological functions of tomato TPK1B RELATED KINASES TPK09 and TPK07.</p><p dir="ltr">To determine the function of TPK09 and TPK07, mutant alleles of these two RLCKs were generated through CRISPR-Cas9 gene editing. Loss of function mutants of <i>tpk09</i> exhibited increased susceptibility to <i>Botrytis</i> but showed no altered responses to the bacterial pathogen <i>P. syringae</i>. Plants carrying mutant alleles displayed reduced immune gene expression and impaired accumulation of reactive oxygen species in response to chitin and flg22. In addition to <i>Botrytis</i> and several plant hormones, the expression of <i>TPK09</i> gene is induced by light but suppressed by darkness. The exposure of wildtype tomato plants to light-emitting diodes (LEDs) reduced hypocotyl length but <i>tpk09</i> mutants were insensitive. Furthermore, <i>tpk09</i> mutants also exhibited increased accumulation of H₂O₂ and extensive necrosis, suggesting a disturbance in cellular homeostasis in response to changes in light spectra, ultimately leading to enhanced susceptibility to <i>Botrytis</i>. Although the global impact of TPK09 on <i>Botrytis</i> induced transcriptome was limited, the expression of the tomato negative regulator of cell death SlBI-like1 gene was significantly reduced in the mutant, particularly in response to the combined effect of <i>Botrytis</i> and LED light. The data suggest TPK09 regulates SlBI-like1 gene expression, but the mechanism is unclear. Further, the impairment of the light stress response in <i>tpk09</i> mutants was substantiated by a reduction in chlorophyll content and damage to the photosynthetic machinery, along with a clear reduction in the expression of genes related to light harvesting and photosynthesis. Regulatory network analysis using RNA-seq data identified TPK09 regulated genes related to stress and oxidative damage. This was further supported from proteomic studies of tomato TPK09-HA transgenic plants. Immunoprecipitation coupled with mass spectrometry revealed that proteins associated with photosynthesis and photosystem subunits were enriched in TPK09 overexpressing plant. Our data establishes that TPK09 is required for fungal resistance and light stress by maintaining functional photosynthetic machinery and ROS homeostasis.</p><p dir="ltr">We also studied the function of tomato TPK07 which is another member of the RLCK-VII family. Through analysis of <i>tpk07</i> mutants, we show that TPK07 it required for resistance to both <i>Botrytis</i> and <i>P. syringae</i> expressing the cysteine protease type III effector AvrPphB which cleaved TPK07 protein. However, further studies are needed to understand the biological function of this cleavage. The expression of TPK07 was induced by <i>Botrytis</i> and the bacterial PAMPs flg22, flg28, and csp22. TPK07 belongs to the RLCK VII-5 subfamily and clusters in the same clade with Arabidopsis PBL34 (AT5G15080), PBL35 (AT3G01300), and PBL36 (AT3G28690) that functions in immunity to <i>P. syringae</i>. In addition, <i>tpk07</i> mutants showed reduced accumulation of ROS in response to chitin and lipopolysaccharide. Moreover, TPK07 is a plasma membrane-localized kinase with a unique N-terminal sequence, offering a research avenue for future structural analysis to understand its contribution in pathogen responses.</p><p dir="ltr">In the last chapter, we conducted comparative analyses of <i>Botrytis</i> induced transcriptome of Arabidopsis and tomato to explain the observed differences between the two plant species in their resistance to <i>Botrytis</i>. Over the years, we observed tomato is more susceptible to <i>Botrytis</i> than Arabidopsis under the same disease assay conditions, but the mechanism is not known. We sought to gain insight into the immune responses of the two species and identify targets for future functional analyses that can explain the differential pathogen responses. Many separate studies<i> </i>have demonstrated<i> </i>that <i>Botrytis</i> causes extensive transcriptional reprogramming in Arabidopsis and tomato. Our results from the comparative studies of in-house generated data identified differentially expressed genes (DEGs), Gene Ontology terms, and metabolic pathways that are specific or shared between the two species. Interestingly, some genes show distinct expression pattern in tomato and Arabidopsis consistent with previous observation that some genes in the two species show contrasting disease resistance functions. Genes with distinct expression patterns warrant further studies to better understand differences in host immune responses between the two species. In addition, transcription factors (TFs) and regulatory hub genes that could be important for further studies were identified.</p><p dir="ltr">In sum, our data establishes the functions of two tomato RLCKs in fungal resistance, interactions between fungal resistance and plant responses to light, and the conserved and contrasting gene expression profiles of tomato and Arabidopsis genes, laying the foundation for future studies.</p>

Cytoplasmic dynein is a microtubule-dependent motor protein which participates in numerous cellular processes. The motor complex consists of two heavy chains, intermediate, light intermediate and 3 families of light chains. Dynein is able to bind to these accessory chains as well as to regulatory proteins which enables the motor protein to fulfil such a variety of cellular processes. The associated light chains participate in long-distance organelle and vesicle transport in interphase and in chromosome segregation during mitosis. However, how these light chains control the activity of the motor protein is still unknown. In this study, I combine molecular genetics and live cell imaging to elucidate the role of the associated dynein light intermediate and light chains in dynein behaviour and early endosome (EE) motility in hyphal interphase cells as well as the anchorage of dynein to the microtubule (MT) plus-end in interphase and mitotic cells. I show that the dynein light intermediate chain (DLIC) as well as the light chain 2 (DLC2, Roadblock) are involved in dynein processivity and EE movement in interphase. The downregulation of either protein results in short hyphal growth which could be caused by a decreased runlength of EE and dynein. In addition, both proteins participate in dynein anchorage to the microtubule plus-end in interphase and mitosis as well as in spindle elongation during mitosis. Each protein causes a decrease of the motor protein dynein at MT plus-ends. Surprisingly, I found only minor or no defects in LC8 or Tctex mutants in the observed functions of dynein. LC8 seems to affect the dynein but not the EE runlength. In this case, dynein is still able to move into the bipolar MT array from where kinesin3 is able to take over EEs and move them towards the cell center. In contrast, Tctex has no effect on dynein or EE runlength or any other observed dynein function in hyphal cells. However, it causes a reduction in spindle elongation. Taken together, DLIC and DLC2 are important for dynein behaviour in long distance transport as well as in spindle positioning and elongation during mitosis. Furthermore, I studied the involvement of the dynein regulators Lis1 and NudE as well as the plus-end binding protein Clip1 (Clip-170 homologue) in the anchorage of dynein to the astral microtubule plus-ends during mitosis. The disruption of the anchorage complex at the astral MT plus-end causes a decrease in dynein number at this site and therefore slower spindle elongation in Anaphase B. Taken together, all three proteins are involved in anchorage of dynein to the astral microtubule tip and the subsequent spindle elongation. Furthermore, these findings also show that Ustilago maydis evolved two different mechanisms to anchor the motor protein to MT plus-ends in hyphal and mitotic cells. The plus-end binding protein Peb1 (EB1 homologue) and the dynein regulator dynactin mediate the dynein anchorage in hyphal cells whereas in mitotic cells the plus-ends binding protein Clip1 and the dynein regulators Lis1 and NudE anchor dynein to astral MT plus-ends.

On retrouve dans le complexe Chrosomus eos-neogaeus une forme cybride ayant le génome nucléaire de C. eos et le génome mitochondrial de C. neogaeus. Ce modèle particulier fournit une occasion unique d’étudier l’influence d’une mitochondrie exogène sur le métabolisme et la physiologie d'organismes vivant en milieu naturel, et s'étant donc adaptés à cette situation cellulaire atypique. La mitochondrie jouant un rôle fondamental vital, nous nous attendons à ce que la présence d’une mitochondrie exogène chez la forme cybride ait un impact sur l’expression de son génome et du protéome qui en découle. L’objectif de ce projet est d’étudier les différences au niveau protéomique entre des individus C. eos purs (forme sauvage) et des cybrides provenant d'habitats similaires afin de faire ressortir au maximum les différences dues à la présence de mitochondries C. neogaeus chez la forme cybride. Pour ce faire, nous avons comparé les protéomes des formes cybride et sauvage en utilisant l'électrophorèse en deux dimensions. Un sous-groupe de protéines produisant un signal spécifique révélé par l’analyse comparative a été identifié et analysé par spectrométrie de masse (LC/MS). Les résultats indiquent que la présence de mitochondries C. neogaeus chez le cybride influence fortement la régulation génique chez ce dernier. De plus, les protéines identifiées apportent des pistes intéressantes supportant l'hypothèse que la présence de mitochondries C. neogaeus chez le cybride rendrait ce biotype plus résistant au froid que la forme sauvage. / The Chrosomus eos-neogaeus genetic complex regroups different forms of hybrids of these two species, among which a cybrid form, that harbours the nuclear genome of C. eos and the mitochondria of C. neogaeus. This peculiar model is thus a unique opportunity to study the influence of an exogenous mitochondria on the metabolism and cellular physiology in a living animal in the wild, and thus perfectly adapted to this atypical cellular environment. Mitochondria being at the core of fundamental biological processes, we expect that the presence of foreign mitochondria will modify gene expression and the resulting proteome of these fishes. The overall goal of this master thesis is thus to compare the proteome of pure (wild type) C. eos with the cybrid form sampled in similar lakes from the same geographical area so that most differences could be attributed to the different mitochondrial genomes. To achieve this goal, we used two dimensional electrophoresis. We selected a sub-group of proteins that showed the most extreme expression differences and identified these spots by mass spectrometric analyses (LC/MS). Results demonstrate that C. neogaeus mitochondria has a strong influence on gene expression in cybrid. Proteins identified bring new clues supporting the hypothesis that cybrid are more cold tolerant than the wild type biotype.

O Fator estimulador de colônias de granulócitos humano recombinante (rhG-CSF) produzido em Escherichia coli é uma proteína não glicosilada com 175 aminoácidos, de grande importância clínica para o tratamento de neutropenias. O presente trabalho propõe a construção de dois sistemas de expressão em E. coli, um sistema para obtenção do rhG-CSF no citoplasma e outro para secreção da proteína recombinante no meio de cultura utilizando a sequência sinal da L-asparaginase II. Os dois sistemas de expressão foram testados e comparados. A partir desses dados, passou-se para as etapas de obtenção do rhG-CSF com o sistema de expressão sem a sequência sinal. As etapas de renaturação e purificação foram eficientes obtendo-se uma proteína com adequado grau de pureza, integridade estrutural e atividade biológica. Essa proteína também foi utilizada com sucesso para a produção de anticorpos policlonais em camundongos. Com os resultados obtidos, a proteína rhG-CSF mostrou-se viável para estudos posteriores em bioreatores e produção em escala-piloto. / The recombinant human granulocyte colony stimulating factor (rhG-CSF) is a non-glycosylated protein with 175 amino acids. This factor plays an important role in hematopoietic cell proliferation and has been widely used for treating neutropenia. The purpose of this work is to construct two expression systems in E. coli; a system for obtaining rhG-CSF in the cytoplasm and the other for secretion of recombinant protein in the culture medium using the signal sequence of L-asparaginase II. The two expression systems were tested and compared. From these data, the next steps for obtaining the rhG-CSF were done with the expression system without the signal sequence. The refolding and purification steps were efficient, resulting in a protein with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of polyclonal antibodies in mice. With these results, the protein rhG-CSF was feasible for further studies in bioreactors and pilot scale production.

Die intrazellulären Parasiten der Bakteriengattung Wolbachia sind weit verbreitet im Phylum der Arthropoden. In vielen Wirten lösen sie eine Paarungsinkompatibilität zwischen nicht infizierten Weibchen und infizierten Männchen aus. Die mögliche Rolle dieser zytoplasmatischen Inkompatibilität in Artbildungsprozessen der Wirtsorganismen wird seit langer Zeit diskutiert. In dieser Arbeit analysieren wir häufig angeführte Kritikpunkte einer solchen Rolle mit Hilfe von mathematischen Modellen, in denen Infektionsdynamik von Wolbachia und Populationsgenetik der Wirte kombiniert werden. Die einzelnen Teile befassen sich mit dem Folgenden: (i) Wir untersuchen die Stabilität von Infektionsmustern in Wirts-Metapopulationen, indem wir kritische Migrationsraten herleiten. (ii) Zur Abschätzung des Einflusses der zytoplasmatischen Inkompatibilität auf den Genfluss zwischen Populationen berechnen wir effektive Migrationsraten. (iii) Wir bestimmen die Bedingungen, die die Verstärkung von Reproduktionsbarrieren durch die Evolution von weiblichen Paarungspräferenzen begünstigen. Schließlich (iv) wenden wir unsere Modelle auf einen realen Artbildungsprozess zweier Drosophila-Arten in Nordamerika an, diskutieren auftretende Probleme und unterbreiten Vorschläge für weiterführende Forschung. Zusammenfassend implizieren unsere Ergebnisse, dass Wolbachien häufig mit der Entstehung neuer Wirtsarten verknüpft sein können, allerdings in den meisten Fällen nur, indem sie als einer von mehreren Faktoren zur reproduktiven Isolation beitragen. Eine Verstärkung sexueller Isolation wird nur unter speziellen Bedingungen bewirkt. / The intracellular bacterial parasites of the genus Wolbachia are widespread among arthropod species. In many hosts, they induce a reproductive incompatibility between uninfected females and infected males. The potential role of this cytoplasmic incompatibility in speciation processes of the bacteria''s hosts has long been debated. In this thesis, we analyze common criticisms of such a role by means of mathematical models, combining Wolbachia infection dynamics and host population genetics. In particular, we are concerned with the following: (i) In order to measure the stability of infection patterns within host metapopulations, we derive critical migration rates. (ii) We evaluate the impact of cytoplasmic incompatibility on gene flow between populations by calculating effective migration rates. (iii) We determine the conditions that favor the evolution of female mating preferences through reinforcement. Finally, (iv) we apply our models to a particular real-world speciation process of two sibling Drosophila species in North America, discuss emerging problems, and suggest future directions of research. In summary, our results implicate that Wolbachia might be a frequent factor in host speciation, but usually only by contributing to overall reproductive isolation among other factors. Reinforcement of premating isolation is selected for only under stringent conditions.