Phospholipase C enzymes are a class of enzymes that catalyse the cleavage of the membrane phospholipid, phosphatidylinositol bisphosphate (PtdIns(4,5)P₂) into the second messengers, inositol trisphosphate (Ins(4,5)P₃) and diacylglycerol (DAG). Six classes of PLC enzymes have been identified based on their structure and mechanism of activation. PLCηs are the most recently identified family and consist of two isozymes, PLCη1 and PLCη2. The aim of this thesis is to further understand the mechanisms of PLCη activation, the role of PLCη2 in relation to neuritogenesis and their roles in certain disease states. Both isoforms were found to be activated by physiological concentrations of intracellular Ca²⁺. Activation of PLCη2 by Gß₁γ₂ was confirmed using a bacterial 2A co-expression system to allow expression of PLCη2, Gß₁ and Gγ₂ with a single plasmid. Localisation studies show a nuclear distribution for PLCη2, but a cytoplasmic distribution for PLCη1 in a neuroblastoma cells line (Neuro2A). PLCη2 has been implicated in brain development and neurite formation. Building on this, a neuronal differentiation model using RA-treated Neuro2A cells stably expressing mutant forms of PLCη2 was utilised, revealing that PLCη2 activity is essential for neuritogenesis but that this process is independent of the enzymes high sensitivity towards Ca²⁺. Furthermore, the direct interaction of PLCη2 and LIMK-1, a previously identified PLCη2 associated protein, is confirmed in the aforementioned neuronal model. Due to the high sensitivity of PLCη enzymes to Ca²⁺ and because of their presence within neurons, they may be involved in Ca²⁺ dysregulation that occurs in certain diseases such as Alzheimer's disease (AD). The role of PLCη2 was assessed in amyloid-ß (Aß) treated differentiated Neuro2A cells, a cellular model for AD pathogenesis. Also a developmental role for PLCη1 was investigated due to a recently identified PLCη1 polymorphism in patients with holoprosencephaly, an embryonic midline defect.

A urocortina 2 (Ucn2) é um peptídeo que pertence à família dos fatores liberadores de corticotrofina (CRF) e, assim como seu receptor específico CRF2R? (corticotropin releasing factor receptor type 2?), encontram-se expressos no músculo esquelético. Embora tenha sido demonstrado que o tratamento sistêmico com Ucn2 seja capaz de induzir hipertrofia e prevenir a perda de massa muscular, nada se conhece acerca dos mecanismos moleculares através dos quais a Ucn2 desempenha suas ações biológicas. O principal objetivo deste trabalho foi investigar o mecanismo de ação da Ucn2 no músculo esquelético de roedores para o aparecimento da resposta hipertrófica e a possível participação das vias de sinalização Akt/mTOR, Akt/Foxo e ERK1/2 neste efeito anabólico. Para isto foi utilizado o modelo de transfecção in vivo da Ucn2 pelo método da eletroporação em músculos tibialis anterior de camundongos. Nestes músculos foram quantificados: 1) o estado de fosforilação de componentes efetores destas vias; 2) moléculas sinalizadoras da via autofágica; 3) a taxa de síntese proteica in vivo e 4) a expressão de genes relacionados à atrofia muscular (atrogenes). Outra metodologia utilizada para verificar o efeito direto da Ucn2 na musculatura esquelética foi a incubação in vitro de músculos soleus e EDL isolados de roedores com este peptídeo a fim de investigar a taxa de degradação proteica total, bem como a atividade dos sistemas proteolíticos lisossomal¸ ubiquitina-proteassoma e dependente de Ca+2. A superexpressão in vivo da Ucn2 por 14 dias promoveu hipertrofia e preveniu a atrofia em músculos tibialis anterior de camundongos normais e submetidos ao modelo de desnervação motora isquiática.Resumo Este crescimento muscular induzido pela Ucn2 in vivo foi associado a ativação das vias de sinalização AMPc/PKA/CREB, AMPc/Epac, Akt/mTOR/S6, Akt/mTOR/4E-BP1 e ERK1/2/eIF4E com consequente estimulação da síntese proteica. Em concordância, utilizando uma técnica de manipulação genética in vivo, demonstramos que a hipertrofia promovida pela Ucn2 é dependente da estimulação das cascatas de sinalização ativadas por Akt e ERK1/2. Ademais, essa alteração fenotípica promovida pela Ucn2 induziu melhora da resistência à fadiga muscular, sendo este impacto funcional dependentente de ERK1/2, mas não de Akt. Além disso, a superexpressão da Ucn2 induziu \"shifting\" para o tipo de fibra oxidativa (tipo I), sendo esta plasticidade possivelmente mediada por PGC-1?, o que pode ter contribuído pelo menos em parte, para o efeito benéfico promovido pela Ucn2 na função muscular. O efeito antiatrófico da Ucn2 in vivo foi associado à estimulação da via Akt/Foxo1,3 concomitante com a redução da atividade transcricional de Foxo, resultando na diminuição da expressão da E3-ligase atrogin-1 e do gene autofágico LC3. Em paralelo, a Ucn2 in vivo promoveu inibição do fluxo autofágico, inferido pelo acúmulo das proteínas LC3-I, LC3-II e p62 nestes músculos. Corroborando os achados in vivo, os efeitos antiproteolíticos da Ucn2 in vitro parecem ser mediados pelo AMPc e envolvem a supressão da atividade do sistema lisossomal/autofágico em músculos EDL de ratos normais. Portanto, além da participação de efetores dowsntream do AMPc, como PKA e EPAC, diferentes quinases participam dos efeitos biológicos da Ucn2 na musculatura esquelética. Esses resultados são importantes para caracterizar novas estratégias terapêuticas capazes de atuar no combate à atrofia muscular em diversas situações catabólicas. / Urocortin 2 (Ucn2) is a peptide that belongs to corticotrophin releasing factors (CRF) family and, as well as its specific receptor CRF2R? (corticotropin releasing factor receptor type 2?), are expressed in skeletal muscle. Although it has been demonstrated that Ucn2 systemic treatment is able to induce hypertrophy and prevent loss of muscle mass, nothing is known about the molecular mechanisms through which Ucn2 plays its biological actions. The main objective of this work was to investigate the Ucn2 mechanism of action in rodent skeletal muscle for the appearance of the hypertrophic response and the possible participation of Akt/mTOR, Akt/Foxo and ERK1/2 signaling pathways in this anabolic effect. For this, an in vivo transfection model of Ucn2 was used by the electroporation method in tibialis anterior muscles of mice. Were quantified in these muscles: 1) the phosphorylation state of effector components of these pathways; 2) signaling molecules of the autophagic pathway; 3) the rate of protein synthesis in vivo and 4) the expression of genes related to muscle atrophy (atrogenes). Another methodology used to verify the direct effect of Ucn2 in skeletal muscle was the incubation of soleus and EDL muscles isolated from rodents with this peptide in vitro in order to investigate the total rate of protein degradation, as well as the activity of the lysosomal, ubiquitin-proteasome and Ca+2 dependent proteolytic systems. Ucn2 overexpression in vivo for 14 days promoted hypertrophy and prevented atrophy in tibialis anterior muscles of normal mice and submitted to the sciatic motor denervation model. This muscle growth induced by Ucn2 in vivo was associated with the activation ofAbstract cAMP/PKA/CREB, cAMP/Epac, Akt/mTOR/S6, Akt/mTOR/4E-BP1 and ERK1/2/eIF4E signaling pathways with consequent stimulation of protein synthesis. In agreement, using a genetic manipulation technique in vivo, we demonstrated that the hypertrophy promoted by Ucn2 is dependent on the stimulation of the signaling cascades activated by Akt and ERK1/2. In addition, this phenotypic alteration promoted by Ucn2 induced an improvement in muscle fatigue resistance, being this functional impact dependent on ERK1/2, but not on Akt. Moreover, Ucn2 overexpression in vivo induced the shift to type I oxidative fiber, and this plasticity is possibly mediated by PGC-1?, which may have contributed at least in part to the beneficial effect promoted by Ucn2 in muscle function. The anti-atrophic effect of Ucn2 in vivo was associated with the stimulation of Akt/Foxo1,3 pathway concomitant with the reduction of Foxo transcriptional activity, resulting in a decrease in the expression of the atrogin-1 E3-ligase and of the autophagic gene LC3. In parallel, Ucn2 in vivo promoted inhibition of autophagic flow, inferred by the accumulation of LC3-I, LC3-II and p62 proteins in these muscles. Corroborating the in vivo findings, the antiproteolytic effects of Ucn2 in vitro appear to be mediated by cAMP and involve the suppression of lysosomal/autophagic system activity in EDL muscles of normal rats. Thus, in addition to the participation of cAMP dowsntream effectors, such as PKA and EPAC, different kinases participate in the biological effects of Ucn2 on skeletal muscle. These results are important to characterize new therapeutic strategies able to prevent muscular atrophy in several catabolic situations.

abstract: Intracellular voltage recordings from single neurons in vitro and in vivo have been fundamental to our understanding of neuronal function. Conventional electrodes and associated positioning systems for intracellular recording in vivo are large and bulky, which has largely restricted their use to single-channel recording from anesthetized animals. Further, intracellular recordings are very cumbersome, requiring a high degree of skill not readily achieved in a typical laboratory. This dissertation presents a robotic, head-mountable, MEMS (Micro-Electro-Mechanical Systems) based intracellular recording system to overcome the above limitations associated with form-factor, scalability and highly skilled and tedious manual operations required for intracellular recordings. This system combines three distinct technologies: 1) novel microscale, polycrystalline silicon-based electrode for intracellular recording, 2) electrothermal microactuators for precise microscale navigation of the electrode and 3) closed-loop control algorithm for autonomous movement and positioning of electrode inside single neurons. First, two distinct designs of polysilicon-based microscale electrodes were fabricated and tested for intracellular recordings. In the first approach, tips of polysilicon microelectrodes were milled to nanoscale dimensions (<300 nm) using focused ion beam (FIB) to develop polysilicon nanoelectrodes. Polysilicon nanoelectrodes recorded >1.5 mV amplitude, positive-going action potentials and synaptic potentials from neurons in the abdominal ganglion of Aplysia Californica. In the second approach, polysilicon microelectrodes were integrated with miniaturized glass micropipettes filled with electrolyte to fabricate glass-polysilicon microelectrodes. These electrodes consistently recorded high fidelity intracellular potentials from neurons in the abdominal ganglion of Aplysia Californica (Resting Potentials < -35 mV, Action Potentials > 60 mV) as well as the rat motor cortex (Resting Potentials < -50 mV). Next, glass-polysilicon microelectrodes were coupled with microscale electrothermal actuators and controller for autonomous intracellular recordings from single neurons in the abdominal ganglion. Consistent resting potentials (< -35 mV) and action potentials (> 60 mV) were recorded after each successful penetration attempt with the controller and microactuated glass-polysilicon microelectrodes. The success rate of penetration and quality of recordings achieved using electrothermal microactuators were comparable to that of conventional positioning systems. Finally, the feasibility of this miniaturized system to obtain intracellular recordings from single neurons in the motor cortex of rats in vivo is also demonstrated. The MEMS-based system offers significant advantages: 1) reduction in overall size for potential use in behaving animals, 2) scalable approach to potentially realize multi-channel recordings and 3) a viable method to fully automate measurement of intracellular recordings. / Dissertation/Thesis / Doctoral Dissertation Biomedical Engineering 2018

Neste trabalho apresentamos um modelo teórico que busca descrever aspectos do movimento bidirecional apresentado por objetos intracelulares (vesículas, organelas, vírus etc, aos quais iremos nos referir simplesmente como (\"vesículas\"), observado, sobretudo em experimentos in vivo. Este movimento nao-difusivo e caracterizado por inversões rápidas em sua direção e é capaz de gerar gradientes de concentração do objeto transportado. Os fenômenos de transporte intracelular são sabidamente mediados por proteínas motoras (como as kinesinas e dinenas) cujo movimento unidirecional sobre _lamentos protéicos e bem caracterizado (kinesinas se movem em direção a extremidade mais enquanto as dinenas se movem em direção a extremidade-menos dos microtúbulos) e é normalmente entendido através de modelos estocásticos que descrevem o comportamento de uma partícula browniana na presença de um potencial assimétrico que varia no tempo (ver Astumian [26], Adjari e Prost [22], Magnasco [23]). Mais recentemente, surgiram na literatura trabalhos que tentam descrever o movimento de partículas motoras interagentes, uma vez que se percebeu que efeitos coletivos que surgem nestas situações podem ser relevantes para os fenômenos de transporte sobre microtúbulos. Uma abordagem para a descrição do comportamento destes sistemas de partículas motoras interagentes é aquela baseada nos modelos para os sistemas difusivos dirigidos\". Em particular, a versão contínua dos modelos do tipo totally asymmetric exclusion processes\" (TASEP) e asymmetric exclusion processes\" (ASEP) tem sido utilizada para o estudo do comportamento da densidade de motores sobre os microtúbulos, através da analise de soluções estacionarias da equação de Burgers correspondente (Parmeggiani et al. [33]). Até agora, entretanto, não existem na literatura tentativas de abordar, com estes modelos, o transporte bidirecional de vesículas mediado por estes motores interagentes. A idéia que apresentamos aqui é associar este estranho tipo de movimento ao movimento de ondas de choque presentes nas soluções transientes da equa_c~ao de Burgers para algumas condições iniciais. Deste modo, as vesículas acompanhando (\"surfando\") os choques fariam o papel de suas correspondentes microscópicas partículas de segunda classe\", introduzidas h_a um bom tempo na literatura [36], [37], [38] para o estudo da dinâmica microscópica dos choques que estão presentes também na versão discreta dos modelos TASEP e ASEP. Neste sentido, é natural que as condições iniciais consideradas, que seriam perturbações no estado estacionário das partículas, possam ser causadas, no sistema real, pela própria interação com a vesícula. É o caso, portanto, de se propor que a geometria deste objeto tenha um papel importante na determinação da direcional de seu próprio movimento no meio intracelular. Esta parece ser, por exemplo, uma alternativa interessante para explicar aspectos do movimento de vírus no interior das células. / In this work we present a theoretical model to describe aspects of the bidirectional movement performed by intracellular structures (vesicles, organelles, viruses etc, to which we refer here simply as \"vesicles\"), observed essentially at in vivo experiments. This nondifusive movement is characterized by rapid inversions in direction and is capable of creating concentration gradients of the transported cargo. The phenomenon of intracellular transport is known to be mediated by motor proteins (such as kinesins and dyneins) whose own unidirectional motion along protein laments is well characterized (kinesins moves to the plus-end direction while dyneins moves to the minus-end direction of the microtubules) and is usually modeled by a stochastic dynamics describing the behavior of a Brownian particle in the presence of a time dependent asymmetrical potential held (see Astumian [26], Adjari and Prost [22], Magnasco [23]). More recently, it appeared in the literature works attempting to describe the movement of interacting motor proteins, since it was realized that collective e_ects emerging from this situation may be relevant to the transport phenomena along microtubules. An approach to describe the behavior of such interacting motor particles is based on existing models for \\driven di_usive systems\". In particular, the continuum versions of the totally asymmetric exclusion processes\" (TASEP) or the asymmetric exclusion processes\" (ASEP) have been used to study the behavior of motors density along microtubules by analyzing the steady state solutions to the corresponding Burgers equation (Parmeggiani et al. [33]). Up to now, however, there are no attempts in the literature to approach in this context the questions related to the bidirecionality of vesicles transported by these interacting motors. The idea we present here is to associate this odd movement to the movement of shock waves presented by the transient solutions of Burgers equation for certain initial conditions. Accordingly, the vesicles accompanying (sur_ng) the shocks fronts would play the role of their microscopic analogous \\particles of second class\" introduced long ago in the literature [36], [37], [38] to study the kinetics of the shocks that are also present in the discrete versions of the TASEP and ASEP. In this regard, it is natural to think that the considered initial conditions, namely perturbations to the motor density with respect to a steady state, can be created in the real systems simply by the interaction with the vesicle. It might then be the case also to propose that the geometry of the vesicle plays an important role to direct its own movement within intracellular environment. This seems to be, for example, an attractive alternative for explaining aspects of virus movement inside the cell.

Intracellular cargo transport involves the movement of critical cellular components (e.g. vesicles, organelles, mRNA, chromosomes) along cytoskeletal tracks by tiny molecular motors. Myosin Va motors have been demonstrated to play a vital role in the transport of cargos destined for the cell membrane by navigating their cargos through the three-dimensional actin networks of the cell. Transport of cargo through these networks presents many challenges, including directional and physical obstacles which teams of myosin Va-bound to a single cargo must overcome. Specifically, myosin Va motors are presented with numerous actin-actin intersections and dense networks of filaments which can act as a physical barrier to transport. Due to the complexities of studying myosin Va cargo transport in cells, much effort has been focused on the in vitro observation and analysis of myosin Va transport along single actin filaments or simple actin cytoskeletal models. However, these model systems often rely on non-physiological cargos (e.g. beads, quantum dots) and two-dimensional arrangements of actin attached to glass surfaces. Interestingly, a disconnect exists between the transport of cargo on these simple model systems and studies of myosin Va transport on suspended 3D actin arrangements or cellular networks which show longer run lengths, increased velocities, and straighter, more directed trajectories. One solution to this discrepancy is that the cell may use the fluidity of the cargo surface, the recruitment of myosin Va motor teams, and the 3D geometry of the actin, to finely tune the transport of intracellular cargo depending on cellular need. To understand how myosin Va motors transport their cargo through 3D networks of actin, we investigated myosin Va motor ensembles transporting fluorescent 350 nm lipid-bilayer cargo through arrangements of suspended 3D actin filaments. This was accomplished using single molecule fluorescent imaging, three-dimensional super resolution Stochastic Optical Reconstruction Microscopy (STORM), optical tweezers, and in silico modeling. We found that when moving along 3D actin filaments, myosin motors could be recruited from across the fluid lipid cargo’s surface to the filaments which enabled dynamic teams to be formed and explore the full actin filaments binding landscape. When navigating 3D actin-actin intersections these teams capable of maneuvering their cargo through the intersection in a way that encouraged the vesicles to continue straight rather than switch filaments and turn at the intersection. We hypothesized that this finding may be the source of the relatively straight directed runs by myosin Va-bound cargo observed in living cells. To test this, we designed 3D actin networks where the vesicles interacted with 2-6 actin filaments simultaneously. Actin forms polarized filaments, which, in cells, generally have their plus-ends facing the exterior of the cell; the same direction in which myosin Va walks. We found that to maintain straight directed trajectories and not become stationary within the network, vesicles needed to move along filaments with a bias in their polarity. This allows for cargo-bound motors to align their motion along the polarized networks and produced productive motion despite physical and directional obstacles. Together this work demonstrates the physical properties of the cargo, the geometric arrangement of the actin, and the mechanical properties of the motor are all critical aspects of a robust myosin Va transport system.

Obligate intracellular pathogens must invade host cells to survive and pose a global health risk. As such, internalization is a critical life stage and represents an excellent therapeutic target. Oxidoreductase exploitation is a thematic invasion strategy among obligate intracellular pathogens. Delineating the mechanisms and proteins mediating this exploitation could identify novel therapeutic targets for many important pathogens. Anaplasma phagocytophilum infects neutrophils by an incompletely defined mechanism, resulting in the emerging potentially fatal disease, human granulocytic anaplasmosis. The bacterial adhesin, Asp14, contributes to invasion by virtue of its C-terminus engaging an unknown receptor. Yeast two-hybrid analysis identified protein disulfide isomerase (PDI) as a putative Asp14 binding partner. Co-immunoprecipitation confirmed this interaction and identified the Asp14 C-terminus as critical to it. PDI reductase activity inhibition impaired bacterial infection of, but not binding to, host cells. A. phagocytophilum failed to productively infect myeloid-specific PDI conditional knock-out mice. This is the first demonstration of microbial PDI exploitation in vivo. Infection of PDI inhibited cells was rescued when bacterial, but not host surfaces were reduced with the reducing agent tris(2-carboxyethyl)phosphine (TCEP). Furthermore, TCEP restored bacterial infectivity after Asp14 inhibition using an antibody that reduces infection. Mutational analyses identified Asp14 residues critical for binding PDI. These data demonstrate that Asp14 binds and brings PDI to disulfide bonds within A. phagocytophilum surface protein(s) that it reduces, enabling infection. Targeting the Asp14 C-terminus could benefit approaches to prevent/treat granulocytic anaplasmosis. A similar approach would identify proteins from other obligate intracellular pathogens that could prove to be protective targets.

AU COURS DE LA THESE, L'ETUDE DU TRANSCRIPTOME DE SOUCHES GARDEL ET SENEGAL VIRULENTES ET ATTENUEES D'E. RUMINANTIUMA ETE REALISEE. UNE ANALYSE DU TRANSCRIPTOME A DIFFERENTS STADES DE DEVELOPPEMENT, A D'ABORD ETE EFFECTUEE POUR LA SOUCHE GARDEL VIRULENTE. AU STADE CORPS RETICULE (FORME INTRACELLULAIRE NON INFECTIEUSE), UNE SUREXPRESSION DES GENES CODANT POUR DES PROTEINES IMPLIQUEES DANS LE METABOLISME, LE TRANSPORT ET L'ECHANGE DE NUTRIMENTS ET DANS LA RESISTANCE AU STRESS OXYDATIF ETAIT OBSERVEE. IL SEMBLERAIT QUEE. RUMINANTIUMMETTE EN PLACE UN PANEL DE MECANISMES POUR SA SURVIE ET SON DEVELOPPEMENT A L'INTERIEUR DE LA CELLULE HOTE. AU STADE CORPS ELEMENTAIRE (FORME EXTRACELLULAlRE INFECTIEUSE), LE GENE DKSA CODANT POUR UN FACTEUR DE TRANSCRIPTION ETAIT SUREXPRIME. CE GENE A ETE MONTRE COMME ETANT IMPLIQUE DANS LA REGULATION DE FACTEURS DE VIRULENCE. IL SEMBLERAIT . DONC, QU'AU STADE CORPS ELEMENTAIRE, IL Y AIT UNE INDUCTION DE MECANISMES DE VIRULENCE. LA COMPARAISON DE L'EXPRESSION DES GENES AU STADE CORPS ELEMENTAIRE ENTRE SOUCHES VIRULENTES ET ATTENUEES A AUSSI ETE EFFECTUEE. NOS RESULTATS ONT MONTRE UNE MODIFICATION IMPORTANTE DE LA MEMBRANE POUR LES SOUCHES VIRULENTES ET ATTENUEES. POUR LES SOUCHES ATTENUEES, IL A ETE MONTRE UNE SUREXPRESSION DES GENES IMPLIQUES DANS LA BIOGENESE MEMBRANAlRE ET UNE SOUS-EXPRESSION·DES PROTEINES DE LA FAMILLE MULTIGENIQUE MAP. CES RESULTATS SUGGERENT QUE LES PROTEINES MAP JOUENT UN ROLE DE LEURRE VIS-A-VIS DE LA REPONSE IMMUNITAIRE PROTECTRICE. DES PROTEINES MEMBRANAlRES HYPOTHETIQUES SONT SUREXPRIMEES A LA FOIS CHEZ LES SOUCHES VIRULENTES ET ATTENUEES. CERTAINES D'ENTRE ELLES SUREXPRIMEES CHEZ LES SOUCHES ATTENUEES SEMBLENT ETRE DE BONS CANDIDATS VACCINAUX ET DEVRAIENT ETRE ETUDIEES / Transcriptomic study of gardel and senegal both virulent and attenuated e. ruminantium strains was conducted during my phd. an analysis of transcriptome at different stages of development has been first conducted for virulent gardel strain. at reticulate body stage (intracellular form non-infectious), over-expression of genes coding for proteins involved in metabolism, transport and exchange of nutrients and resistance to oxidative stress was observed. at this stage of development, e. ruminantium seems to activate mechanisms for its survival and development within the host cell. at elementary body stage, dksa the gene encoding for a transcription factor was over-expressed. this gene has been shown to be involved in the regulation of virulence factors. it seems, therefore, at the elementary body stage, e. ruminantium induces its virulence factors. secondly, we compare the transcriptome of elementary body between virulent and attenuated strains. our results showed an important membrane modification of attenuated and virulent strains. for attenuated strains, we observed an over-expression of genes involved in membrane biogenesis and a diminution of expression of map multigenic family. it seems that map proteins subvert the protective immune response. hypothetical membrane proteins are over-expressed in both virulent and attenuated strains. some over-expressed proteins in attenuated strains seem to be good vaccine candidates and willstudied.

Selenium is an essential trace mineral of fundamental importance to human health. Its beneficial functions are largely attributed to its presence within a group of proteins named selenoproteins in the form of the amino acid selenocysteine (Sec). Recently, it was revealed that the human selenoproteome consists of 25 selenoproteins, and for many of them their function remains unknown. The most prominent known roles of selenoproteins are to maintain the intracellular redox homeostasis, redox regulation of intracellular signalling and thyroid hormone metabolism. Sec incorporation into selenoproteins employs a unique mechanism that involves decoding of the UGA stop codon. The process requires interplay between distinct, intrinsic features such as the Sec Insertion Sequence (SECIS) element, the tRNASec and multiple protein factors. The work presented in this thesis has focused on characterising the regulation of human SECIS binding protein 2, SBP2, a factor central to this process. Experimental approaches combined with bioinformatics analysis revealed that SBP2 is subjected to alternative splicing. A total of nine alternatively spliced transcripts appear to be expressed in cells, potentially encoding five different protein isoforms. The alternative splicing events are restricted to the 5?-region, which is proposed to be dispensable for Sec incorporation. One of the variants identified, contains a mitochondrial targeting sequence that was capable of targetting SBP2 into the mitochondrial compartment. This isoform also appears to be expressed endogenously within the mitochondria in cells. Previous reports have depicted SBP2 as a ribosomal protein, despite the presence of a putative Nuclear Localisation Signal (NLS). In this study it was found that SBP2 subcellular localisation is not restricted to ribosomes. Intrinsic functional NLS and Nuclear Export Signals (NESs), enable SBP2 to shuttle between the nucleus and the cytoplasm via the CRM1 pathway. In addition, the subcellular localisation of SBP2 appears to play an important role in regulating Sec incorporation into selenoproteins. The subcellular localisation of SBP2 is altered by conditions imposing oxidative stress. Several oxidising agents induce the nuclear accumulation of SBP2, which occurs via oxidation of cysteine residues within a novel redox-sensitive cysteine rich domain (CRD). Cysteine residues were to form disulfide bonds and glutathione-mixed disulfides during oxidising conditions, which are efficiently reversed in vitro by the thioredoxin and glutaredoxin systems, respectively. These modifications negatively regulate selenoprotein synthesis. Cells depleted of SBP2 are more sensitive to oxidative stress than control cells, which correlated with a substantial decrease in selenoprotein synthesis after treatment with oxidising agents. These results provide direct evidence that SBP2 is required for Sec incorporation in vivo and suggest that nuclear sequestration of SBP2 under such conditions may represent a mechanism to regulate the expression of selenoproteins. Collectively, these results suggest that SBP2 is regulated at multiple levels: by alternative splicing, changes in subcellar localisation and redox control.

<p>The protein degradation process is of vital importance for the cell to maintain cellular functions. An important enzyme in this process is the multimeric tripeptidyl-peptidase II (TPP II). It removes tripeptides from a free N-terminus of the substrates. TPP II has broad substrate specificity and wide-spread distribution, suggesting that the TPP II gene is a house-keeping gene. However, the levels of both mRNA and TPP II protein varies during different conditions and the TPP II gene promoter was therefore identified and characterized. It is a 215 bp fragment just upstream of the coding sequence. This fragment lacks a TATA-box but contains an initiator, two inverted CCAAT-boxes and an E-box. The CCAAT-boxes and the E-box were found to bind the nuclear factor Y (NF-Y) and upstream stimulatory factor-1 (USF-1) respectively. The CCAAT-boxes appear to be most important for the transcriptional activation. Furthermore, several silencer element were identified further upstream of the 215 bp promoter and the octamer binding factor Oct-1 was found to bind one of these fragments. If Oct-1 is responsible for the inhibition of the transcription of the TPP II gene remains to be investigated. In addition, the substrate specificity was investigated. For this purpose an expression system using <i>Pichia pastoris</i> was developed. The purified recombinant TPP II was found to have the same enzymatic properties as the native enzyme. In order to identify the amino acids involved in the binding of the N-terminus of the substrate, wild-type murine TPP II and four mutants E305Q, E305K, E331Q and E331K were purified. Steady-state kinetic analysis clearly demonstrated that both Glu-305 and Glu-331 are important for this binding as the K_M^app is more than 10² higher for the mutants than wild-type. Finally, the pH-dependence for cleavage of two chromogenic substrates was compared for TPP II from different species.</p>

Thelohania solenopsae are intracellular pathogens found in the red imported fire ant, Solenopsis invicta. These pathogens cause detrimental effects to their fire ant hosts. The present study revealed that the midgut and the meconium materials from pupating fourth instar larvae were possible vehicles for the horizontal transmission of the disease. The pathogen was further found to cause a reduction of humeral proteins. In SDS-PAGE stained with silver, several proteins were observed only in controls but not in infected fire ant queens. Different queens were found to have variable proteins reduced due to infection of this pathogen. Furthermore, vitellogenin titers were found to be significantly reduced in infected fire ant queens, although the infection rates of the fat body cells was found to be less than 20%. Finally, although the pathogens did not directly induce apoptosis in infected cells, there were more infected cells undergoing apoptosis than uninfected cells. There was no evidence to support the idea that infected fat body cells became more resistant to apoptosis inducers.