Alopecia areata (AA) is a chronic inflammatory disease of hair follicles manifesting as patchy areas of hair loss on the scalp and body. Development of AA is associated with pen- and intra-follicular inflammation of anagen stage hair follicles, primarily by CD4+ and CD8+ cells. We hypothesized that if cell-mediated cytotoxicy against hair follicles is to be a component of the hair loss disease mechanism, increased expression of genes and products typical of cytotoxic cells, as well as increased apoptosis activity within affected hair follicles, would be expected to occur in the lesional skin compared to the normal skin. Furthermore, we studied gene expression levels of multiple cytokines and characteristic chemokines, using the C3FI/HeJ mouse model of AA. mRNA expression levels of granzyme A, granzyme B, perform Fas, Fas ligand, TNF-cL, TNF-aRl and R2, TRAIL, TRAILR, TRAMP, Thi-, Th2-, and Th17-associated cytokines, as well as multiple chemokines were compared between the skin, draining lymph nodes, thymus and spleens of normal and AA-affected mice using quantitative reverse transcriptase PCR. FasL, granzyme A, granzyme B, pro- and anti-inflammatory cytokines were all highly up-regulated in the skin of AA-affected mice. Immunohistochemical studies of the skin revealed that, although greater numbers of granzyme B and FasL expressing cells were present in AA affected skin, the cells were morphologically diffusely distributed and not exclusively located within the focal pen- and intrafollicular infiltrate. The majority of these cells were further characterized as mast cells, which were also found in substantially greater numbers in the skin of mice with AA compared to their normal haired controls. Almost no perform expressing cells were identified in AA affected mouse skin and TUNEL staining suggested relatively limited apoptosis activity in hair follicle keratinocytes. In conclusion, while granzymes and FasL may play important roles in disease development, the profiles and patterns of expression are not consistent with direct cell-mediated cytotoxic action against the follicular epithelium in chronic mouse AA. Potentially, hair growth inhibiting cytokines may play a more dominant role in AA development than previously thought. Furthermore, mast cells, with their increased presence around hair follicles in the AA affected mouse skin and their ability to express granzyme B and FasL, are suggested as potential key players in the pathogenesis of AA.

Denna rapport sammanfattar det examensarbete som utförts 2012 på kandidatnivå av Erika Dahlström och Josefin Hult på Mälardalens Högskola i Eskilstuna. Examensarbetet har utförts i samarbete med ÅF Technology AB i Västerås. Projektets idé uppstod i ett tidigare fiktivt högskoleprojekt där tanken om ett vindkraftverk skulle vara intressant för privatpersoner kom till. Detta projekt har vidareutvecklat denna tanke och projektet har då utvecklats till att syfta till att undersöka om det finns en marknad för miniverk i byggsatsform avsett för privatpersoner, vad marknaden efterfrågar samt utveckla och konstruera en miniverksmast. Ett miniverk är ett vindkraftverk som vanligtvis inte behöver bygglov. För att kunna besvara hur marknaden ser ut har omfattande research utförts samt en marknadsundersökning. Från researchen sågs att det finns liknande befintliga produkter vilket innebar att projektets syfte gick från att konceptutveckla ett helt miniverk till att differentiera vår produkt från konkurrenternas. Detta gjordes genom att utveckla en mast som ger brukaren större valmöjligheter genom bland annat underlätta montering samt underhåll av sitt miniverk. Vid konstruktion har fokus lagts på knäckningsberäkningar då knäckning måste tas på största allvar för att undvika potentiellt allvarliga olyckor. Beräkningar har jämförts med simuleringar utförda i Solid Works vilket visar på att beräkningarna bör vara korrekta. Resultatet blev en 15,4 meter hög mast uppdelad i fem sektioner. Masten höjs och sänks genom en vev, block och vajrar. För att säkra att masten står stabilt finns åtta stycken stabiliserande vajrar fästa på två mastsektioner. Denna mast uppfyller majoriteten av de krav som ställts upp i en kravspecifikation. Att alla krav inte kunde uppfyllas beror på projektets begränsade tid. Dock har tydliga rekommendationer getts för att möjliggöra en vidareutveckling av detta resultat så att alla krav kan uppfyllas. Under hela produktutvecklingsprocessen har ett flertal olika produktutvecklingsverktyg använts som stöd och kvalitetssäkring i arbetet. Arbetet med projektet har fungerat väl, dock var en del frågor inom projektet mycket komplexa vilket innebar en del förseningar.

<p>Lymph nodes are organs of efficiency. Once activated, they essentially function to optimize and accelerate the production of the adaptive immune response, which has the potential to determine survival of the host during an initial infection and protect against repeated infections, should specific and appropriate immunological memory be sufficiently induced. We now have an understanding of the fundamental structure of lymph nodes and many of the interactions that occur within them throughout this process. Yet, lymph nodes are dynamic and malleable organs and much remains to be investigated with regards to their responses to various types of challenges. In this work, we examined multiple inflammatory scenarios and sought to understand the complex ways that lymph nodes can be externally targeted to impact immunity. First, we outline a novel mechanism of cellular communication, where cytokine messages from the periphery are delivered to draining lymph nodes during inflammation. These signals are sent as particles, released by mast cells, and demonstrate the ability of the infected tissue to communicate to lymph nodes and shape their responses. Based on these interactions, we also explored the ability to therapeutically or prophylactically modulate lymph node function, using bioengineered particles based on mast cell granules, containing encapsulated cytokines. When we used these particles as a vaccine adjuvant, we were able to polarize adaptive immune responses, such as to promote a Th1 phenotype, or enhance a specific attribute of the immune response, such as the production of high avidity antibodies. We then explore three examples of lymph node-targeting pathogens: Salmonella typhimurium, Yersinia pestis and Dengue virus. Each of these pathogens has a well-characterized lifecycle including colonization of draining lymph node tissue. In the case of S. typhimurim, we report that the virulence this pathogen depends on a specific shut down of the chemotactic signals in the lymph node that are required to maintain appropriate cellular localization within it. Our results demonstrate that these architecture changes allow S. typhimurim to target the adaptive immune process in lymph nodes and contribute to its spread in vivo and lethality to the host. With Y. pestis, similar targeting of cellular trafficking pathways occurs through the modulation of chemokine expression. Y. pestis appears to use the host's cellular trafficking pathways to spread to lymph nodes in two distinct waves, first exploiting dendritic cell movement to lymph nodes and then enhancing monocyte chemoattractants to replicate within monocytes in draining lymph nodes. These processes also promote bacterial spread in vivo and we further demonstrate that blocking monocyte chemotaxis can prolong the host's survival. In the third example of pathogen challenge, we report for the first time that mast cells can contribute functionally to immunosurveillance for viral pathogen, here, promoting cellular trafficking of innate immune cells, including NK cells, and limiting the spread of virus to draining lymph nodes. For each of these three examples of lymph node targeting by microbial pathogens, we provide data that modulation of cellular trafficking to and within lymph nodes can drastically influence the nature of the adaptive immune response and, therefore, the appropriateness of that response for meeting a unique infectious challenge. Cumulatively this work highlights that a balance exists between host and pathogen-driven modulation of lymph nodes, a key aspect of which is movement of cells within and into this organ. Cytokine and chemokine pathways are an area of vulnerability for the host when faced with host-adapted pathogens, yet the lymph node's underlying plasticity and the observation that slight modulations can be beneficial or detrimental to immunity also suggests the targeting of these pathways with therapeutic intentions and during vaccine design.</p> / Dissertation

Understanding energetic potential of habitat patches is important for management designed to provide adequate habitat for wildlife species. Great Smoky Mountains National Park (GSMNP) has a high density of black bears that have been studied intensively from 1968-1997; habitats within the Park are relatively undisturbed, and similar vegetative cover types can be found throughout the southern Appalachian mountains. Black bear reproduction in the Park has been correlated to hard mast production, however little work has been done to assess the importance of soft mast. Geographic Information System (GIS) based habitat use models have been developed for bears in the Park, yet the importance of foods in determining habitat selection, and the possibility of sexual habitat segregation due to food availability have not yet been determined. The primary objectives of the study included estimation of the location, timing, and amount of caloric production by 19 important black bear foods and determination of the significance of caloric production by mast type, season, overstory vegetation type, and plant species. Secondary objectives were to test for correlation of bear habitat use with estimated caloric production from mast, and to test for sexual segregation of habitats based on caloric production. This study was limited to the northwest quadrant of GSMNP during 1995.

Incidences of fungal infections are on the rise in immunosuppressed people. Predominant causative agents for these mycoses are species of the genus Candida, including Candida albicans, Candida glabrata and Candida dublieniensis. Despite a wide range of emerging pathogens, C. albicans remains the leading cause. According to recent epidemiological studies, blood stream infections with C. albicans cause annually ~55% mortality in approximately 300,000 patients from intensive care units worldwide. Furthermore, the percentage of morbidity linked to oral, esophageal and vulvovaginal mycoses cause by C. albicans reach up to 90%. Reasons for these medical concerns are the lack of efficient diagnostics and antifungal therapy. Here, we therefore sought to find novel antifungal strategies inspired by innate immune cells, such as neutrophils. These phagocytes are able to block the fungal pathogenicity. Neutrophils are bloodstream leukocytes serving as the first line of defense against pathogenic microbes. It has been shown that neutrophils have a strong antifungal activity by impairing the conversion of the dimorphic C. albicans from yeast to hyphal form (Y-H). Consequently, we raised the question whether other immune cells, such as mast cells, with less phagocytic cabapilities may have similar activity to neutrophils. Mast cells are tissue-dwelling cells. Mucosal tissue is rich in mast cells and usually constitutes the entry ports for fungal pathogens into the human body. A contribution of mast cells in antifungal defense is, thus, very likely. We human explored mast cell functions upon encounter with fungal pathogens. Interestingly, human mast cells show a transient potential to impair fungal viability. To understand the mechanism behind this impairment we analyzed the human mast cell functions in more detail. We found that human mast cells challenged with C. albicans, immediately degranulate and secrete distinct cytokines and chemokines in an orchestrated manner. The chemokines secreted attract neutrophils. Mast cells moreover are able to internalize fungal cells and to ‘commit suicide’ by releasing extracellular DNA traps that ensnare the pathogen.   The effectiveness of future antifungals is depended on targeting the pathogen virulence with more efficiency. The dimorphism of C. albicans is proven to be essential its virulence. Blockage of this switching ability could render the pathogen avirulent. Consequently, we screened for compounds that mimic the neutrophils anti-dimorphic activity by screening small chemical molecule libraries that block Y-H transition. The screening of big chemical libraries requires a reliable, reproducible and rapid high-throughput screening assay (HTS). We developed an HTS assay based on automated microscopy and image analysis, thereby allowing to distinguish between yeast and filamentous forms. In order to find the ideal Y-H blocker, we also evaluated the cell viability via the count of ATP levels when challenged with the respective small chemical molecules.   Drug development is an elaborate and expensive process. We therefore applied our screening setup to identify antidimorphic/antifungal activity in compounds from two different chemical libraries including FDA-approved drugs. The study disclosed 7 off-patent antifungal drugs that have potent antimycotic activity, including 4 neoplastic agents, 2 antipsychotic drugs and 1 antianemic medication. In a nutshell, we aimed to mimic the anti-dimorphic/antifungal activity of neutrophils with small chemical molecules. Furthermore, we elucidated how immune cells contribute to antifungal defense to exploit these mechanisms for the development of novel antifungal therapies. Thus, this thesis provides novel tools for the discovery of more efficient compounds, identifies previously unknown antifungal aspect of off-patent FDA-approved drugs and highlights the interplay of mast cells with pathogenic fungi with the aim to define new screening strategies.

Alopecia areata (AA) is a chronic inflammatory disease of hair follicles manifesting as patchy areas of hair loss on the scalp and body. Development of AA is associated with pen- and intra-follicular inflammation of anagen stage hair follicles, primarily by CD4+ and CD8+ cells. We hypothesized that if cell-mediated cytotoxicy against hair follicles is to be a component of the hair loss disease mechanism, increased expression of genes and products typical of cytotoxic cells, as well as increased apoptosis activity within affected hair follicles, would be expected to occur in the lesional skin compared to the normal skin. Furthermore, we studied gene expression levels of multiple cytokines and characteristic chemokines, using the C3FI/HeJ mouse model of AA. mRNA expression levels of granzyme A, granzyme B, perform Fas, Fas ligand, TNF-cL, TNF-aRl and R2, TRAIL, TRAILR, TRAMP, Thi-, Th2-, and Th17-associated cytokines, as well as multiple chemokines were compared between the skin, draining lymph nodes, thymus and spleens of normal and AA-affected mice using quantitative reverse transcriptase PCR. FasL, granzyme A, granzyme B, pro- and anti-inflammatory cytokines were all highly up-regulated in the skin of AA-affected mice. Immunohistochemical studies of the skin revealed that, although greater numbers of granzyme B and FasL expressing cells were present in AA affected skin, the cells were morphologically diffusely distributed and not exclusively located within the focal pen- and intrafollicular infiltrate. The majority of these cells were further characterized as mast cells, which were also found in substantially greater numbers in the skin of mice with AA compared to their normal haired controls. Almost no perform expressing cells were identified in AA affected mouse skin and TUNEL staining suggested relatively limited apoptosis activity in hair follicle keratinocytes. In conclusion, while granzymes and FasL may play important roles in disease development, the profiles and patterns of expression are not consistent with direct cell-mediated cytotoxic action against the follicular epithelium in chronic mouse AA. Potentially, hair growth inhibiting cytokines may play a more dominant role in AA development than previously thought. Furthermore, mast cells, with their increased presence around hair follicles in the AA affected mouse skin and their ability to express granzyme B and FasL, are suggested as potential key players in the pathogenesis of AA.

Disertacijoje sprendžiama telekomunikacinių stiebų efektyvumo problema. Tyrimo objektas – statiškai apkrauti naujos konstrukcijos stiebai su kombinuotomis atotampomis. Darbe išanalizuota šiuolaikinių stiebų konstrukcinės formos ir skaičiavimo metodai. Nagrinėjama sukurta stiebo su kombinuotomis atotampomis konstrukcija, kurioje vietoj įprastųjų siūloma taikyti kombinuotas atotampas. Disertacijoje šiems stiebams skaičiuoti pateikta inžinerinė skaičiavimo metodika. Pateikti naujo stiebo, veikiamo statinių apkrovų, elgsenos analizės skaitinių ir eksperimentinių laboratorinių tyrimų rezultatai. Stiebo su kombinuotomis atotampomis kamieno lenkiamiesiems momentams reguliuoti darbe siūloma taikyti parengtą stiebų komponavimo metodiką. Šių stiebų efektyvumui nustatyti pateikti gretinamieji tyrimai su įprastos sandaros stiebais. Disertaciją sudaro bendroji darbo charakteristika, 5 skyriai, pagrindiniai darbo rezultatai ir išvados, literatūros sąrašas. / Тhe dissertation deals with the problem of telecommunication masts efficiency. The subject of research – masts of a new structure with combined guys under static loads. The study deals with constructive forms and analysis methods of up-to-date masts. It analyses into a created structure of the mast, wherein it is suggested to apply combine guys instead of the ordinary ones. Engineering guyed masts with combined guys static analysis method is given this research. The numerical and experimental laboratory assay of a new mast under static loads behaviour is given. It is proposed in the study to apply a worked out composing method of masts with combined guys to control the bending moments of shaft. Compared research results are given to measure efficiency of these masts against masts of an ordinary structure. The dissertation includes a general characteristic of study, five chapters, main results and conclusions, list of references.

Тhe dissertation deals with the problem of telecommunication masts efficiency. The subject of research – masts of a new structure with combined guys under static loads. The study deals with constructive forms and analysis methods of up-to-date masts. It analyses into a created structure of the mast, wherein it is suggested to apply combine guys instead of the ordinary ones. Engineering guyed masts with combined guys static analysis method is given this research. The numerical and experimental laboratory assay of a new mast under static loads behaviour is given. It is proposed in the study to apply a worked out composing method of masts with combined guys to control the bending moments of shaft. Compared research results are given to measure efficiency of these masts against masts of an ordinary structure. The dissertation includes a general characteristic of study, five chapters, main results and conclusions, list of references. / Disertacijoje sprendžiama telekomunikacinių stiebų efektyvumo problema. Tyrimo objektas – statiškai apkrauti naujos konstrukcijos stiebai su kombinuotomis atotampomis. Darbe išanalizuota šiuolaikinių stiebų konstrukcinės formos ir skaičiavimo metodai. Nagrinėjama sukurta stiebo su kombinuotomis atotampomis konstrukcija, kurioje vietoj įprastųjų siūloma taikyti kombinuotas atotampas. Disertacijoje šiems stiebams skaičiuoti pateikta inžinerinė skaičiavimo metodika. Pateikti naujo stiebo, veikiamo statinių apkrovų, elgsenos analizės skaitinių ir eksperimentinių laboratorinių tyrimų rezultatai. Stiebo su kombinuotomis atotampomis kamieno lenkiamiesiems momentams reguliuoti darbe siūloma taikyti parengtą stiebų komponavimo metodiką. Šių stiebų efektyvumui nustatyti pateikti gretinamieji tyrimai su įprastos sandaros stiebais. Disertaciją sudaro bendroji darbo charakteristika, 5 skyriai, pagrindiniai darbo rezultatai ir išvados, literatūros sąrašas.

Oral tolerance is the state of immunologic non-responsiveness that is established following oral antigen consumption. Failures of oral tolerance can result in food allergy. The mechanisms regulating oral tolerance are not well understood, but similar mechanisms may control tolerance to foods and commensal microbes in the intestine. The specific roles of many pattern recognition receptors (PRRs) and innate cells have not been examined in the context of oral tolerance. Mast cells are innate sentinel cells positioned at mucosal surfaces, and have been identified as key regulators of peripheral tolerance to allografts. Toll-like receptor 2 (TLR2) is a PRR involved in bacterial responses and the regulation of intestinal inflammation. We evaluated the impact of mast cells, TLR2, immunoglobulin E (IgE)-mediated mast cell activation, TLR2 activation, and histamine receptor blockade in the development of oral tolerance in mice. Models of tolerance to ovalbumin, peanut butter, and cow’s milk were established. Oral tolerance was assessed in wild type, TLR2-deficient, or mast cell-deficient mice and was measured primarily by analysis of antigen-specific antibody levels after a systemic antigen challenge. The development of antigen-specific Tregs was also assessed. We observed that neither mast cells nor TLR2 were necessary for oral tolerance induction. Moreover, IgE-mediated mast cell activation and antihistamine treatment did not significantly alter oral tolerance induction. TLR2 activators, notably Pam3CSK4, were administered orally concurrent with food antigen and were found to impair oral tolerance to a later systemic antigen challenge. When Pam3CSK4 was administered as an oral adjuvant with ovalbumin, a profound selective enhancement of the IgA response to oral challenge was observed. These results highlight an important differential regulation of oral tolerance by TLR2. Oral TLR2 activation selectively promotes IgA responses to antigen upon repeated oral challenge but prevents the maintenance of oral tolerance upon a systemic challenge. Taken together these results suggest that mast cells are not essential regulators of oral tolerance, but TLR2 is involved in regulating IgA and IgE responses during oral and systemic challenges. These findings inform mechanisms of commensal tolerance and have implications for the potential therapeutic manipulation of oral tolerance to foods.

<p>Urinary tract infections (UTIs) are the second most common type of infection identified in the clinical setting and disproportionately afflict women. UTIs most frequently manifest in the form of infection of the lower urinary tract, involving the bladder. Uropathogens, particularly uropathogenic E. coli, progressively colonize the urethra and ascend to the bladder, where they initiate cystitis. In some cases, infection further ascends through the ureters and reaches the kidneys, where it causes pyelonephritis. Infection of both the upper and lower urinary tract can have serious ramifications for the host, and this is in large part due not to infection itself but to host-directed responses to bacterial insults. </p><p> In this thesis, I will describe and discuss two distinct aspects of UTIs. In the first study, in vivo work in a mouse model of urinary tract infection revealed a novel role for mast cells, which are tissue-resident granulated innate immune cells, in directing the detachment and death of epithelial cells during cystitis, facilitating the clearance of bacteria from the bladder. An ex vivo porcine bladder infection model suggested a specific role for mast cell granules and the proteases contained therein, which was corroborated with in vitro experiments utlizing isolated mast cell granules and human epithelial cells to demonstrate granule-induced exfoliation and cell death. From this work, it is clear that mast cells play a highly targeted role in modulating urothelial integrity during bladder infection by mediating host-directed epithelial loss.</p><p> In the second study described in this dissertation, the synergistic roles of both pyelonephritis and vesico-ureteric reflux (VUR), a congenital urinary tract defect that results in the improper backflow of urine from the bladder to the kidney, in the development of reflux nephropathy, a fibrotic host response characterized by renal scar formation, were elucidated in a series of in vivo experiments. Specifically, the C3H mouse, which is naturally susceptible to VUR, was utilized to characterize the dynamics of kidney infection and the onset of reflux nephropathy. Renal scarring was dependent on the presence of sustained kidney infection and the accompanying inflammatory response due to VUR, while neither transient infection nor reflux alone were sufficient to provoke nephropathy. Thus, the development of reflux nephropathy is dependent upon the confluence of both infection and VUR. </p><p> This body of work reveals the double-edged sword of the host inflammatory response to urinary tract infection. In the bladder, mast cell activation and degranulation leads to granule-induced epithelial exfoliation and consequently a reduction in the bacterial burden in the bladder. However, the sustained inflammatory response that accompanies pyelonephritis in vesico-ureteric reflux-affected individuals results in significant damage to the kidney without any accompanying reduction in infection. These findings highlight the dueling roles of the host inflammatory response to infection in the upper and lower urinary tract and strongly suggest that differential clinical approaches to cystitis and pyelonephritis are necessary to promote an effective mast cell in the bladder in the former and facilitate the clearance of renal infection while mitigating tissue damage in the latter.</p> / Dissertation