The Hypothalamic Pituitary Adrenal (HPA) axis is the coordinator of adaptive responses to physical and psychological stress. The central nervous system plays a key role in modulation of both basal and adaptive HPA axis functions. In fact, since long ago, animal studies have shown that acute and chronic exposure to glucocorticoids (a stress hormone released due to HPA axis activation, cortisol in humans) affects the function and the morphology of brain areas such as the hippocampus and the cingulate cortex. This thesis is based on novel neuroimaging methodologies used to investigate the interactions of psychological stress, cortisol and the brain. It consists of three functional studies and a morphometric one. In the first functional study we show that the hippocampus (where glucocorticoid receptors are most abundant) plays a role in initiation of an HPA axis stress response. In the second study, we provide evidence that besides hippocampus, the neural activity in the so-called "default mode network" (DMN), especially the anterior cingulate cortex (ACC), relates to interindividual variations in HPA axis response to psychological stress. In the third study we have investigated the cortisol-modulation of the DMN. Again, we provide evidence for a role of the ACC and the orbitofrontal cortex in negative feedback inhibition of the HPA axis activity. Finally, we show a morphological link between the ACC and the cortisol response to awakening which is an index of basal HPA axis activity. Overall, our findings confirm the critical role of the ACC and mesolimbic system in HPA axis regulation. These findings also draw attention to the interactions between functional subregions of the medial prefrontal cortex and states of HPA axis function prior to stress onset---suggesting an interplay of the monitoring and the executive planning roles of the medial prefrontal cortex in behavioral adaptation to stress. Beyond stress research, our findings offer a framework for combining neuroimaging and neuroendocrinology to better understand the interindividual variances in behavior, and perhaps to better identify subgroups at risk of psychological disorders.

High plasma concentrations of Lipoprotein(a) (Lp(a)) have been identified as a risk factor for a variety of atherogenic disorders such as cerebrovascular disease, peripheral vascular disease, and coronary heart disease. Lp(a) consists of a lipoprotein moiety containing apolipoproteinB-100 (apoB-100), as well as apolipoprotein(a) (apo(a)), a unique glycoprotein to which the majority of Lp(a) functions are attributed. Variation in the number of identically repeated kringle IV type 2 (KIV2) motifs of apo(a) forms the molecular basis of Lp(a) isoform size heterogeneity, which is a hallmark of this lipoprotein. There is a general inverse correlation between apo(a) size and plasma Lp(a) concentrations, attributed in part to less efficient secretion of larger apo(a) isoforms from hepatic cells. The present study provides a preliminary investigation into processes involved in apo(a) secretion, with respect to isoform size, to understand this inverse correlation at a molecular level. Pulse-chase experiments were performed in human embryonic kidney (HEK 293) cells and human hepatoma (HepG2) cells, both stably expressing differently-sized recombinant apo(a) isoforms representing the range of apo(a) sizes observed in the population. The folding kinetics for the different apo(a) isoforms were determined by changes in the mobility of the non-reduced radiolabelled species on SDS-PAGE gels. In HEK 293 cells, the rate at which apo(a) is folded correlated well with isoform size. In HepG2 cells, however, folding times were comparable regardless of isoform size. Apo(a) secretion from both cell lines exhibited size-dependency. Preliminary experimentation on endoplasmic reticulum (ER)-resident protein modifications of apo(a) was performed, resulting in the identification of apo(a) interactions with PDI, Erp57, Calnexin, Grp78, Grp94, and EDEM. Preliminary experiments indicate a role for intracellular apo(a) degradation in the amount of apo(a) that is secreted from HepG2 cells, although an isoform size dependency of this degradation process cannot be established with current experimental data. Further experimentation is required to confirm enzyme interactions with differently-sized apo(a) isoforms, to identify other chaperones involved in apo(a) secretion, and to confirm the role of proteasomes in intracellular apo(a) degradation. This may, in turn, provide information regarding the mechanism of how apo(a) secretion from hepatic cells is regulated. / Thesis (Master, Biochemistry) -- Queen's University, 2009-09-20 19:10:09.497

Tau est une protéine associée aux microtubules enrichie dans l’axone. Dans la maladie d’Alzheimer, Tau devient anormalement hyperphosphorylée, s’accumule dans le compartiment somato-dendritique et s’agrège pour former des enchevêtrements neurofibrillaires (NFTs). Ces NFTs se propagent dans le cerveau dans un ordre bien précis. Ils apparaissent d’abord dans le cortex transenthorinal pour ensuite se propager là où ces neurones projettent, c’est-à-dire au cortex entorhinal. Les NFTs s’étendent ensuite à l’hippocampe puis à différentes régions du cortex et néocortex. De plus, des études récentes ont démontré que la protéine Tau peut être sécrétée par des lignées neuronales et que lorsqu’on injecte des agrégats de Tau dans un cerveau de souris, ceux-ci peuvent pénétrer dans les neurones et induire la pathologie de Tau dans le cerveau. Ces observations ont mené à l’hypothèse que la protéine Tau pathologique pourrait être sécrétée par les neurones, pour ensuite être endocytée par les cellules avoisinantes et ainsi propager la maladie. L’objectif de la présente étude était donc de prouver la sécrétion de la protéine Tau par les neurones et d’identifier par quelle voie elle est secrétée. Nos résultats ont permis de démontrer que la protéine Tau est sécrétée par des neurones corticaux de souris de type sauvage ainsi que dans un modèle de surexpression dans des cellules HeLa et PC12. Nos résultats indiquent que la sécrétion de Tau se ferait par les autophagosomes. Finalement, nous avons démontré que la protéine Tau sécrétée est déphosphorylée et clivée par rapport à la protéine Tau intracellulaire non sécrétée. / Tau, a microtubule-associated protein, is enriched in the axon. In Alzheimer’s disease, Tau becomes hyperphosphorylated, redistributes to the somato-dendritic compartment and forms aggregates called neurofibrillary tangles (NFTs). The NFTs propagates in a predictable manner in particular neuronal networks. Indeed, they appear in the trans-entorhinal region and then propagate to the entorhinal cortex where the trans-entorhinal cortex projects. Then, the NFTs propagate to the hippocampus and to different regions of the cortex and neocortex. Recent studies have reported that Tau can be secreted by neuronal cell lines. Besides, when aggregates of Tau protein were injected in mouse brain, they could enter neurons and induced Tau pathology. Based on those observations, it was speculated that Tau could be secreted by neurons and then captured by neighbouring cells to propagate Tau pathology in the brain. The goal of the present study was to prove that Tau can be secreted by neurons and to find the secretory pathway involved in Tau secretion. Moreover, the phosphorylation state of Tau protein was examined and compared to intracellular non-secreted Tau. Our results showed that Tau is secreted by cortical neurons isolated from wild-type mice and by HeLa and PC12 cells overexpressing human Tau. Our results also indicated that autophagosomes would be involved in Tau secretion. Finally, we found that secreted Tau was dephosphorylated and cleaved compared to the non-secreted intracellular Tau.

Intracellular bacteria have developed various mechanisms to enter and persist in host cells and, at the same time, to evade the host immune response. One such pathogen is Francisella tularensis, the etiological agent of tularemia. After phagocytosis, this Gram-negative bacterium quickly escapes from the phagocytic compartment and replicates in the host cell cytosol. For this mode of infection, several components of the Francisella pathogenicity island (FPI) are critical. Interestingly, some FPI proteins share homology to components of Type VI Secretion Systems (T6SSs), but their assembly and functionality remains to be shown in Francisella.The thesis focused on the characterization of several of these FPI components; more specifically, how they contribute to the infection cycle as well as their possible role in the putative T6SS. We identified three unique mutants, ΔiglG, ΔiglI and ΔpdpE, which to various degrees were able to escape the phagosomal compartment, replicate in the host cytosol and cause host cell cytotoxicity. In contrast, ΔiglE as well as mutants within the conserved core components of T6SSs, VgrG and DotU, were defective for all of these processes. In the case of IglE, which is a lipoprotein and localized to the outer membrane of the bacterial cell wall, residues within its N-terminus were identified to be important for IglE function. Consistent with a suggested role as a trimeric membrane puncturing device, VgrG was found to form multimers. DotU stabilized the inner membrane protein IcmF, in agreement with its function as a core T6SS component. The functionality of the secretion system was shown by the translocation of several FPI proteins into the cytosol of infected macrophages, among them IglE, IglC and VgrG, of which IglE was the most prominently secreted protein. At the same time, the secretion was dependent on the core components VgrG, DotU but also on IglG. Although we and others have shown the importance of FPI proteins for the escape of F. tularensis, it has been difficult to assess their role in the subsequent replication, since mutants that fail to escape never reach the growth-permissive cytosol. For this reason, selected FPI mutants were microinjected into the cytosol of different cell types and their growth compared to their replication upon normal uptake. Our data suggest that not only the metabolic adaptation to the cytosolic compartment is important for the replication of intracytosolic bacteria, but also the mechanism of their uptake as well as the permissiveness of the cytosolic compartment per se.

<p>Due to its close proximity to the gastrointestinal tract, the human urinary tract is</p><p>subjected to constant barrage by gut-­associated bacteria. However, for the most part, this tract has resisted infection by various microbes. The impregnability of the urinary tract to microbial colonization is attributable to the ability of the bladder to promptly sense and mount robust responses to microbial challenge. A powerful mechanism for the elimination of invading bacteria was recently described in bladder epithelial cells, involving non-­lytic ejection of intracellular bacteria back into the extracellular milieu. In spite of the effectiveness of this defense strategy, much of the underlying mechanisms surrounding how this powerful cellular defense activity detects intracellular UPEC and shuttles them from their intracellular location to the plasma membrane of BECs to be exported remains largely a mystery.</p><p> Here, we describe uropathogenic E.coli (UPEC) expelled from infected bladder</p><p>epithelium cells (BECs) within membrane-­bound vesicles as a distinct cellular defense</p><p>response. Examination of the intracellular UPEC revealed that intracellular bacteria were</p><p>initially processed via autophagy, the conventional degradative pathway, then delivered</p><p>into multivesicular bodies (MVBs) and encapsulated in nascent intraluminal vesicle membrane. We further show the bacterial expulsion is triggered when intracellular UPEC follow the natural degradative trafficking pathway and reach lysosomes and attempt to neutralize its pH to avoid degradation. This pathogen-­mediated activity is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized on lysosomes, which spontaneously initiates lysosome exocytosis resulting in expulsion of exosome-­encased bacteria. These studies reveal a cellular default system for lysosome homeostasis and also, how it is coopted by the autonomous defense program to clear recalcitrant pathogens.</p> / Dissertation

Pseudomonas aeruginosa causes various infections in humans, animals and plants. Type III secretion system (T3SS) is one of the essential virulence factors used by P. aeruginosa. In this study, a previously uncharacterized gene PA0466 and its role in T3SS regulation have been examined. The results indicate that PA0466 is a novel T3SS regulator. It regulates T3SS directly through an unknown pathway and has a minor effect on the GacA-RsmA pathway. Besides the role in the interaction between the pathogen and the host, T3SS may also play a role in the interspecies interaction. A real-time PCR based Competitive Index (CI) assay was used to compare the wild type and T3SS mutant with and without the presence of Staphylococcus spp.. The results indicate that PAO1 was more competitive than exsA mutant and the difference was even bigger in the presence of Staphylococcus, suggesting T3SS may play a significant role in bacterium-bacterium interaction.

Ištyrėme dviems šimtams gyvūnų ašarų sekrecijos lygį 400 akių. Tiriamieji objektai – kliniškai sveiki įvairios rūšies, amžiaus, lyties, veislės ir įvairiomis akių ligomis sergantys gyvūnai (triušiai, kiaulės, karvės, arkliai, šunys, katės). Analizavome gyvūno rūšies, amžiaus, veislės, lyties, paros ir metų laikotarpių, bei akių ligų įtaką kiekybiniams ašarų sekrecijos rodikliams, taip pat narkozės įtaką ašarų sekrecijai bendrosios anestezijos protokole naudojant skirtingus bendruosius anestetikus. Darbo tikslas. Nustatyti kai kurių naminių gyvūnų kiekybinius ašarų liaukos sekrecijos rodiklius ir įvertinti kai kurių veiksnių įtaką jiems. Visiems gyvūnams atlikome bendrąjį klinikinį tyrimą ir išmatavome ašarų kiekį akyje Širmerio testu, rodančiu bendrąjį (bazinį ir refleksinį kartu) ašarų sekrecijos lygį, ašarų liaukų sekrecinį pajėgumą. Rezultatai ir išvados. Analizuodami tyrimo rezultatus nustatėme, kad gyvūno rūšis turi įtakos ašarų sekrecijos kiekybiniams parametrams: daugiausia ašarų sekretuoja arklių akių ašarų liaukos – 25,45 ± 1,04 mm/min. Nustatėme, kad gyvūnų lytis daro įtaką ašarų liaukų sekretuojamam ašarų kiekiui ir ji priklauso nuo gyvūno rūšies. Tirtų gyvūnų, išskyrus kates, ašarų sekrecija yra aktyvesnė dieną. Tirtų gyvūnų, išskyrus arklius, ašarų sekrecija yra aktyvesnė vasarą. Gyvūnų ašarų sekrecijos pajėgumas ženkliai sumažėja sergant akių ligomis: konjunktyvitu ir uveitu, o sergant katarakta ašarų sekrecijos pokyčiai yra nežymūs. Narkotizuotų kiaulių akyse... [toliau žr. visą tekstą] / We examined two hundred animal tear secretion rate in 400 eyes. Subjects – various clinically healthy animal species, age, sex, breed, and animals with various eye diseases (rabbits, pigs, cows, horses, dogs, cats). We also analyzed animal specie, age, gender, time of a day, seasons influence as well as the influence to the tears secretion using different general anesthetics. Objective. Set some pet quantitative lacrimal gland secretion rates and assess the factors which influence them. We have done a general clinical examination and measurements of the amount of tears in the eyes for all animals using schirmer test, which shows a general (basic and reflex together) tear secretion level of the lacrimal secretion capacity. Results and conclusions. Analyzing the results we found that animal species affects the secretion of tears quantitative parameters. Most tears secrets horse eye lacrimal glands – 25,45 ± 1,04 mm / min. We found that animals sex affects the tear glands secretion quantity and it depends on the animal species. Of all analyzed animals except cats tear secretion is more active in day time. Of all analyzed animals except horses, tear secretion is more active in the summer. Animal tear secretion capacity is significantly reduced in patients with eye diseases: conjunctivitis and uveitis but patients with cataract tear secretion changes are minor. In narcotized pigs decreased excretion of tears in the eyes also anesthesia with double cocktail of thiopental –... [to full text]

Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins. Conventional purification processes are lengthy, technically complicated, product specific and time-consuming. To address this issue, herein we develop a one step purification system that due to the nature of the non-selective secretion system and the versatility of ion-exchange membrane chromatography can be widely applied to the production of many recombinant proteins. This was achieved through the integration of the intrinsically coupled upstream, midstream and downstream processes, a connection that is rarely exploited. A bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography medium while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange medium, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level of 487 U/L/OD600, with more than 90% was localized in the extracellular medium. Both, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. Furthermore, formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. iv At harvest, the whole non-clarified culture broth was applied directly to a tangential flow filtration anion-exchange membrane chromatography system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry). Due to their size, cells remained in the retentate while the extracellular medium penetrated the membrane. Most contaminate proteins were captured by the anion-exchange membrane, whereas the purified PAC was collected in the filtrate. The batch time for both cultivation and purification was less than 24 h and recombinant PAC with high purity (19 U/mg), process yield (74%), and productivity (41 mg/L) was obtained.

The study of diabetes mellitus is vital in this day and age because its incidence is increasing at an alarming rate. Diabetes results in the loss of function of beta-cells within the pancreas. Insulin resistance contributes to diabetes but the human body can compensate in various ways such as increasing the islet cell mass, glucose disposal and insulin secretion, in order to prevent the onset of diabetes. Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are two integral hormones important in both glucose homeostasis and islet cell growth. Early studies using cultured islet cells have demonstrated positive regulation of beta-cell growth by both GH and IGF-I. To evaluate their relevance on normal beta-cell growth, compensatory growth, as well as in insulin responsiveness, we have used two mouse models that represent opposite manipulations of the GH/IGF-I axis. Specifically, the growth hormone receptor gene deficient (GHR-/-) and the IGF-I overexpression (MT-IGF) mice, to help understand the role of glucose homeostasis and islet cell growth in the GH/IGF-I axis. GH is essential for somatic growth and development as well as maintaining metabolic homeostasis. It is known that GH stimulates normal islet cell growth. Moreover, GH may also participate in islet cell overgrowth and compensate for insulin resistance induced by obesity. To determine whether the islet cell overgrowth is dependent on GH signaling, we studied the response of GHR-/- mice to high-fat diet (HFD)-induced obesity. We also studied the insulin responsiveness in GHR-/- mice. On the other hand, IGF-I promotes embryonic development, postnatal growth and the maturation of various organ systems. The notion that IGF-I stimulates islet cell growth has been challenged in recent years by results from IGF-I and receptor gene targeted models. We have characterized MT-IGF mice which overexpress the IGF-I gene. / The results of our studies indicate that (1) GH is essential for normal islet cell growth, but not required for compensatory overgrowth of the islets in response to obesity, (2) GHR gene deficiency caused delayed insulin responsiveness in skeletal muscle; in contrast to elevated insulin sensitivity in the liver; (3) although overexpression does not stimulate islet cell growth, a chronic IGF-I elevation caused significant hypoglycemia, hypoinsulinemia, and improved glucose tolerance, (4) finally IGF-I overexpression mice are resistant to experimental diabetes.

Diabetes mellitus is a bihormonal disorder with hyperglycemia due to deficiency of insulin and hypersecretion of glucagon. To improve diabetes treatment it is important to clarify the signal transduction of glucagon secretion. The cytoplasmic Ca2+ concentration ([Ca2+]i), an important determinant of hormone secretion, and the membrane potential were recorded in individual mouse α-cells. Glucagon and insulin secretion were measured from mouse islets and glucagon secretion from hamster glucagonoma cells. Glucose inhibited glucagon secretion from islets and glucagonoma cells with maximal effect at 7 mM, indicating a direct action on the α-cells. High concentrations of glucose paradoxically stimulated glucagon secretion. Whereas glucose inhibition of glucagon release was associated with lowering of [Ca2+]i, stimulation of secretion at high glucose concentrations was Ca2+-independent. Adrenaline, which is a potent stimulator of glucagon secretion, increased [Ca2+]i by α1- and β-adrenergic mechanisms involving mobilization of intracellular Ca2+ from the endoplasmic reticulum (ER) and influx of the ion across the plasma membrane. Ca2+ mobilization could be attributed to generation of inositol 1,4,5-trisphosphate and cAMP, and influx occurred through voltage-dependent L-type channels activated by a depolarizing store-operated current. Glucose hyperpolarized the α-cells and inhibited adrenaline-induced [Ca2+]i signalling. At 3 mM, glucose had a pronounced stimulatory effect on Ca2+ sequestration in the ER, shutting off store-operated Ca2+ influx. The α-cells express ATP-regulated K+ channels but pharmacological blockade of these channels neither interfered with the hyperpolarizing and [Ca2+]i lowering effects of glucose nor with the inhibition of glucagon secretion. In contrast, activation of the depolarizing store-operated mechanism prevented glucose-induced, hyperpolarization, lowering of [Ca2+]i and inhibition of glucagon secretion. It is proposed that adrenaline stimulation and glucose inhibition of glucagon release involve modulation of a store-operated depolarizing current. The U-shaped dose response relationship for glucose-regulated glucagon secretion may explain the hyperglucagonemia in diabetes.