Introdução: O gene DMRT1 é um fator muito importante, o qual induz a determinação sexual masculina. Estudos mais recentes têm demonstrado que o Dmrt1 possui um papel significante no desenvolvimento ovariano. Deleções restritas ao gene DMRT1 têm sido raramente identificadas em pacientes com disgenesia gonadal (DG) sem outras características sindrômicas. Objetivo: Pesquisar a presença de haploinsuficiência do gene DMRT1 (deleções e/ou mutações inativadoras) em um grupo grande de pacientes não sindrômicos com distúrbios do desenvolvimento sexual (DDS) por anormalidades gonadais. Polimorfismos do DMRT1, como fatores potenciais pelas anormalidades gonadais, foram também identificados. Pacientes e Métodos: Foram avaliados cerca de 39 pacientes portadores de DDS por anormalidades do desenvolvimento gonadal 46,XY: 24 com disgenesia gonadal parcial e 15 pacientes com disgenesia gonadal completa. As regiões codificadoras do DMRT1 e o domínio DM (exon 1) foram amplificados e sequenciados. A análise de Multiplex ligation probe amplification (MLPA) do DMRT1 foi realizada usando um kit comercial. Resultados: Deleção parcial ou total do DMRT1 não foi identificada pela técnica de MLPA. Oito variantes alélicas do DMRT1 foram identificados. Uma nova variante c.968-15insTTCTCTCT foi identificada em 6,4% e em 14,3% dos alelos dos pacientes 46,XY e indivíduos controles, respectivamente. Conclusão: Este estudo sugere que deleções parciais ou completas no DMRT1 e mutações inativadoras não são frequentemente encontradas em pacientes com anormalidades do desenvolvimento gonadal. Além disso, nenhuma das variantes alélicas identificadas neste grupo de pacientes poderia ser considerada como um marcador potencial polimórfico para disgenesia gonadal / Introduction Dmrt1 gene is a very important factor in inducing male sex determination, and more recently it has been demonstrated that Dmrt1 plays a significant role in ovary development. DMRT1 deletions have rarely been identified in patients with 46,XY gonadal dysgenesis (GD) without syndromic features. Objective- To screen for the presence of DMRT1 haploinsufficiency (deletions and/or inactivating mutations) in a large cohort of non-syndromic patients with disorder of sex development (DSD) due to abnormalities of gonadal development. DMRT1 polymorphisms, as potential susceptibility factors for gonadal abnormalities, were also investigated. Subjects and Methods- We evaluated 39 patients with 46,XY GD: 24 patients with the partial, and 15 with the complete form. The entire coding region (éxons 2-5) of DMRT1 and the DM domain (exon 1) were PCR-amplified and direct sequenced. Multiplex ligation probe amplification (MLPA) analysis of DMRT1 was carried out using a commercial kit. Results- Partial or total deletion of DMRT1 was not identified by MLPA technique. Eight allelic variants of DMRT1 were identified. The novel variant c.968-15insTTCTCTCT was identified in 6.4% and in 14.3% of the alleles of 46,XY patients and control subjects, respectively Conclusion- This study suggest that complete or partial DMRT1 deletions and inactivating mutations are not frequently found in patients with abnormalities of gonadal development. Additionally, none of the allelic variants identified in this cohort of patients could be considered a potential polymorphic susceptibility marker for gonadal dysgenesis

This study of Shelley's intellectual development as it is reflected in these philosophical poems is offered in the hope that knowledge of Shelley's idealism may inspire faith in the beauty which life can possess and trust in the high ideals which alone can create such beauty.

The present study focuses on the Vindelälven-Juhttátahkka Biosphere Reserve (BR) in Northern Sweden, which serves as an implementation site of UNESCO’s Man and the Biosphere (MAB) programme. In response to a series of environmental and social problems identified in the specific locale, the area inhabited by both “Swedish” and “Sami” people, is designed to serve henceforth as a learning site for sustainable development. Taking Michel Foucault’s work on discourse and power as a reference point, in this study we analyze the discourse that permeates life in the specific milieu, to understand how power operates in the BR and look for resistance. Following Foucault’s theorization of discourse, the study has employed Foucauldian Discourse Analysis (F.D.A.) on the empirical material, gathered from official documents and interviews with people in the BR. Our research has concluded that by employing its scientific programme, UNESCO’s discourse has managed to a large extent to construct in this place a reality that does not allow a future to be imagined outside of the context of sustainable development. This study has found its participants to be influenced by UNESCO’s discourse and has constrained them from perceiving present or imagining future realities that do not sustain the power of the Swedish state under the global neoliberal rule. Lastly, our research has illustrated that the participants who have been found to respond to their experiences have also been found to resist the discourse permeating the BR.

Optical sensor systems that integrate Long-Period-Gratings (LPG) as the detection arm have been proven to be highly sensitive and reliable in many applications. With increasing public recognition of threats from bacteria-induced diseases and their potential outbreak among densely populated communities, an intrinsic, low-cost biosensor device that can perform quick and precise identification of the infection type is in high demand to respond to such challenging situations and control the damage those diseases could possibly cause. This dissertation describes the development of a biosensor platform that utilizes polymer thin films, known as ionic self-assembled multilayer (ISAM) films, to be the sensitivity- enhancing medium between an LPG fiber and specific, recognition layer. With the aid of cross- linking reactions, monoclonal antibodies (IgG) or DNA probes are immobilized onto the surface of the ISAM-coated fiber, which form the core component of the biosensor. By immersing such biosensor fiber into a sample suspension, the immobilized antibody molecules will bind the specific antigen and capture the target cells or cell fragments onto the surface of the fiber sensor, resulting in increasing the average thickness of the fiber cladding and changing the refractive index of the cladding. This change occurring at the surface of the fiber results in a decrease of optical power emerging from the LPG section of the fiber. By comparing the transmitted optical power before and after applying the sample suspension, we are able to determine whether or not certain bacterial species have attached to the surface of the fiber, and as a consequence, we are able to determine whether or not the solution contains the targeted bacteria. This platform has the potential for detection of a wide range of bacteria types. In our study, we have primarily investigated the sensitivity and specificity of the biosensor to methicillin- resistant Staphlococcus aureus (MRSA). The data we obtained have shown a sensitive threshold at as low as 102 cfu/ml with pure culture samples. A typical MRSA antibody-based biosensor assay with MRSA sample at this concentration has shown optical power reduction of 21.78%. In a detailed study involving twenty-six bacterial strains possessing the PBP2a protein that enables antibiotic resistance and sixteen strains that do not, the biosensor system was able to correctly identify every sample in pure culture samples at concentration of 104 cfu/ml. Further studies have also been conducted on infected mouse tissues and clinical swab samples from human ears, noses, and skin, and in each case, the system was in full agreement with the results of standard culture tests. However, the system is not yet able to correctly distinguish MRSA and non-MRSA infections in clinical swab samples taken from infected patient wounds. It is proposed that nonspecific binding due to insufficient blocking methods is the key issue. Other bacterial strains, such as Brucella and Francisella tularensis have also been studied using a similar biosensor platform with DNA probes and antibodies, respectively, and the outcomes are also promising. The Brucella DNA biosensor is able to reflect the existence of 3 Brucella strains at 100 cfu/ml with an average of 12.2% signal reduction, while negative control samples at 106cfu/ml generate an average signal reduction of -2.1%. Similarly, the F. tularensis antibodies biosensor has shown a 25.6% signal reduction to LVS strain samples at 100 cfu/ml, while for negative control samples at the same concentration, it only produces a signal reduction of 0.05%. In general, this biosensor platform has demonstrated the potential of detecting a wide range of bacteria in a rapid and relatively inexpensive manner. / Ph. D.

Neisseria gonorrhoeae (Ng) which causes gonorrhea has become multidrug-resistant, necessitating the development of novel therapeutics and vaccines. mAb 2C7 which targets an epitope within an important virulence factor, the lipooligosaccharide (LOS), is a candidate therapeutic mAb. Ninety-four percent of clinical isolates express the 2C7-epitope which is also a vaccine target. Ng expresses multiple LOS(s) due to phase-variation (pv) of LOS glycosyltransferase (lgt) genes. mAb 2C7 reactivity requires a lactose extension from the LOS core Heptose (Hep) II (i.e. lgtG ‘ON’ [G+]). Pv results in HepI with: two (2-), three (3-), four (4-), or five (5-) hexoses (Hex). How HepI glycans impact Ng infectivity and mAb 2C7 function are unknown and form the bases of this dissertation. Using isogenic mutants, I demonstrate that HepI LOS glycans modulate mAb 2C7 binding. mAb 2C7 causes complement (C’)-dependent bacteriolysis of three (2-Hex/G+, 4-Hex/G+, and 5-Hex/G+) of the HepI mutants in vitro. The 3-Hex/G+ mutant (resistant to C’-dependent bacteriolysis) is killed by neutrophils in the presence of mAb and C’. In mice, 2- and 3-Hex/G+ infections are significantly shorter than 4- and 5-Hex/G+ infections. A chimeric mAb 2C7 that hyperactivates C’, attenuates only 4- and 5-Hex/G+ infections. This study enhances understanding of the role of HepI LOS pv in gonococcal infections and shows that longer HepI glycans are necessary for prolonged infections in vivo. This is the first study that predicts in vitro efficacy of mAb 2C7 against all four targetable HepI glycans thereby strengthening the rationale for development of 2C7-epitope based vaccines and therapeutics.

Tumor diseases are severe problem worldwide with increasing number of patients suffering from various types of malignancies. Many of approved therapeutics cause serious side toxicities. Therefore, there are intensive efforts to improve cancer treatment protocols. The aim of this study was to deplete regulatory T (Treg) cells without affecting other immunocompetent cells playing a positive role in tumor eradication. Treg cells were reported to hamper anti-tumor immunity and promote tumor growth and survival. Thus, their selective elimination could lead to induction of anti-tumor responses and tumor rejection if combined with chemotherapy with selected N-(2- hydroxypropyl)methacrylamide (HPMA) copolymer-bound drug conjugates. Original approach was to deplete of Treg cells without the use of anti-CD25 mAb that has been widely exploited for Treg cell elimination; however, its long-term persistence in circulation together with inhibitory effect on activated effector cells (CD25+ ) are its main disadvantages. Thus, Treg cells were sensitized to cell cycle-specific cytostatic drugs via application of IL-2/anti-IL-2 JES6.1 mAb immunocomplexes that induce vigorous selective proliferation of this cell population. Subsequent application of cell cycle-specific cytostatics showed steep decrease of Treg cell...

Monoklonale Antikörper (mAk) werden bereits erfolgreich zur therapeutischen Depletion verschiedener Zellpopulationen in vivo verwendet, die Mechanismen der Depletion sind jedoch unklar geblieben. In dieser Arbeit wurden im Mausmodell die molekularen Grundlagen der CD4+ T-Zelldepletion (CD4 TZD) nach einmaliger Gabe (i.p.) von 100 µg des anti-CD4-mAk YTS191.1 untersucht. Dabei konnte eine starke Korrelation zwischen Depletion und der Modulation des CD4-Moleküls von der Oberfläche beobachtet werden. Gleichzeitig zeigten sich organabhängige Unterschiede, sowohl im zeitlichen Verlauf, als auch in der Effizienz der Depletion. Im Thymus konnten weder Depletion noch Modulation detektiert werden, in Milz und Lymphknoten (Lk) war die CD4 TZD nach starker CD4-Modulation bereits nach 48 h mit 80-90 % maximal, in den Peyer-Plaques jedoch niedriger und verzögert (50-60 % nach 72 h). Anhand C3-defizienter Mäuse konnte ferner kein wesentlicher Beitrag von Komplement an der CD4 TZD beobachtet werden. Im Gegensatz dazu konnte durch die Verwendung verschiedener FcGamma-Rezeptor (FcGammaR)-defizienter Mäuse (FcGammaRI, FcGammaRII, FcGammaRIII, FcGammaRI/III und FcRGamma) wie auch durch die Blockade des FcGammaRIV eine starke, zudem organabhängige Beteiligung von FcGammaR an der CD4 TZD gezeigt werden. Während in der Milz die CD4 TZD von FcGammaRIV vermittelt wurde, waren in den Lk und Peyer-Plaques FcGammaRI/III involviert. Diese Befunde korrelierten mit der starken Expression von FcGammaRIV in Milz, Lunge, Darm, Niere und Leber, während in den Lk nur eine schwache und in Thymus und Peyer-Plaques keine Expression detektiert werden konnte. Innerhalb der Milz konnten erstmalig F4/80hoch Makrophagen als FcGammaRIV+ identifiziert und somit als potenzielle Effektorzellen der CD4 TZD bestimmt werden. Der direkte Vergleich der Depletion von CD4+ T-Zellen mit der Depletion von ICOS+ T-Zellen verdeutlichte darüber hinaus, dass die Effizienz der Zelldepletion nicht nur von den Eigenschaften des verwendeten mAk, sondern auch von denen des Zielmoleküls abhängig ist. / Monoclonal antibodies (mAb) are efficiently used for the therapeutic depletion of various cells in vivo yet the mechanisms of depletion are still unclear. In this work, the molecular principles of CD4+ T cell depletion (CD4 Tcd) by a single application of 100 µg of the anti-CD4 mAb YTS191.1.1 were investigated in the mouse. A strong correlation between the depletion and the surface modulation of the CD4 molecule could be observed. At the same time, organ-dependent differences in the kinetics as well as in the efficiency of depletion could be detected. In the thymus, neither modulation nor depletion were detectable. In the spleen and the lymph nodes (Ln), the modulation was strong and the depletion was maximal (80-90%) 48 h after mAb treatment. Interestingly, both modulation and depletion were decreased and delayed (50-60% after 72 h) in the Peyer`s patches. By using C3-deficient mice, no major contribution of complement to the CD4 Tcd was seen. On the contrary, with the help of different FcGamma-receptor (FcGammaR)-deficient mice (FcGammaRI, FcGammaRII, FcGammaRIII, FcGammaRI/III, and FcRGamma) and through the blockade of FcGammaRIV, a strong organ dependent involvement of FcGammaR could be shown. While the depletion in the spleen was clearly dependent on FcGammaRIV, in the Ln and the Peyer`s patches, FcGammaRI/III were involved. These findings correlated with the strong expression of FcGammaRIV in the spleen, the lung, the colon, the kidney, and the liver, while in the Ln the expression was weak and undetectable in the thymus and the Peyer`s patches. For the first time, F4/80high macrophages in the spleen could be identified as also being FcGammaRIV+, and are therfore considered as the potential effector cells of the CD4 Tcd. The direct comparison of the depletion of T cells via CD4 or ICOS pointed out that the target cell depletion is not only dependent on the properties of the mAb used, but also on those of the target molecule.

Abrin is a potent toxin obtained from the seeds of Abrus precatorius. It is a heterodimeric glycoprotein consisting of an A and a B subunit linked together by a disulfide bond. The toxicity of the protein comes from the A subunit harboring RNA-N-glycosidase activity which cleaves the glycosidic bond between the ribose sugar and the adenine at position 4324 in 28S rRNA. The depurination of a specific adenine residue at position 4324 results in loss of conformation of the 28S rRNA at the α sarcin/ricin loop to which elongation factor-2 (EF-2) binds, during the transloction step of translation, leading to inhibition of protein synthesis. The B subunit of abrin is a galactose specific lectin. The lectin activity enables the toxin to gain entry inside cells on binding to receptors with terminal galactose. After entering cells, a few molecules of abrin reach the endoplasmic reticulum (ER) via the retrograde transport, where the disulfide bond between the A and the B subunits gets cleaved. Then the A chain escapes into the cytosol where it binds to its target, the α-sarcin loop of the 28S ribosomal RNA and inhibits protein synthesis. Apart from inhibition of protein synthesis, exposure of cells to abrin leads to the loss of mitochondrial membrane potential (MMP) resulting in the activation of caspases and finally apoptosis. However, whether apoptosis is dependent on the inhibition of protein synthesis has not been elucidated. The major objectives of this study are therefore to delineate the signaling pathways involved in abrin-induced apoptosis. The thesis is divided into 4 Chapters: Chapter 1. provides a overview of the general properties of RIPs, with a brief history, classification, trafficking and biological activities of the toxins. This chapter also discusses their potential use in bio-warfare and the treatments available for management of toxicity. Chapter 2 and 3 discuss the results obtained on studies aimed at gaining insights into the signaling pathways involved in abrin-induced apoptosis. Chapter 4 focuses on the research carried out to understand the mechanisms of neutralization of abrin by the mAb D6F10. Towards the first objective, chapter 2 elucidates the role of endoplasmic reticulum (ER) stress signaling in abrin-induced apoptosis using the human T-cell line, Jurkat as a model system. It could be concluded that the inhibition of protein synthesis by the catalytic A subunit of abrin could result in accumulation of unfolded proteins in the ER leading to ER stress which triggers the unfolded protein response (UPR) pathway. The ER resident trans-membrane sensors IRE1 (Inositol-requiring enzyme 1), PERK (PKR-like ER kinase) and ATF6 (Activating transcription factor 6) are the important players of UPR in mammalian cells. These sensors inhibit translation and increase the levels of chaperones to restore protein homeostasis. However, if the ER stress is prolonged, apoptotic pathways get activated to remove severely damaged cells in which protein folding defects cannot be resolved. Recent studies have shown that endoplasmic reticulum (ER) stress induces apoptosis by activating initiater caspases such as caspase-2 and -8 which eventually trigger mitochondrial membrane potential loss and activation of downstream effector capases-9 and -3. Phosphorylation of eukaryotic initiation factor 2α and upregulation of CHOP [CAAT/enhancer binding protein (C/EBP) homologous protein], important players involved in ER stress signaling by abrin, suggested activation of ER stress in the cells. ER stress is also known to induce apoptosis via stress kinases such as p38 MAPK and JNK. Activation of both the pathways was observed upon abrin treatment and found to be upstream of the activation of caspases. However, abrin-induced apoptosis was found be dependent on p38 MAPK but not JNK. We also observed that abrin induced activation of caspase-2 and caspase-8 and triggered Bid cleavage leading to mitochondrial membrane potential loss and thus connecting the signaling events from ER stress to mitochondrial death machinery. Few toxins belonging to the family of ribosome inactivating proteins such as Shiga toxin have been observed to induce DNA damage in human endothelial cells and activate p53/ATM-dependent signaling pathway in mammalian cells. To further investigate the role of abrin on activation of DNA damage signaling pathway, we analysed the phosphorylation of H2AX and ATM, which are markers for double strand DNA breaks. We observed phosphorylation of H2AX and ATM upon abrin treatment but not when cells were pretreated with the broad spectrum pan caspase inhibitor. This study suggested that the DNA damage observed was an indirect effect of caspase-activated DNase. We concluded from the studies in chapter 2 that inhibition of protein synthesis by abrin can trigger endoplasmic reticulum stress leading to mitochondria-mediated apoptosis. Further studies were conducted to understand the dependence of ER stress on inhibition of protein synthesis and are presented in chapter 3. For this study, we have used an active site mutant of abrin A chain (R167L) which exhibits lower protein synthesis inhibitory activity than the wild type abrin A chain. Recombinant wild type and mutant abrin A chains were expressed in E.coli and purified. Since, abrin A chain requires the B chain for internalization into cells, both wild type and mutant abrin A chains were conjugated to native ricin B chain to generate a hybrid toxin. Next, we have compared the toxic effects of the two conjugates in cells. The rate of inhibition of protein synthesis mediated by the mutant ricin B-rABRA (R167L) conjugate was slower than that of the wild type ricin B-rABRA conjugate but it could trigger ER stress leading to mitochondrial mediated apoptosis in cells though delayed, suggesting that inhibition of protein synthesis is the major factor contributing to abrin-mediated apoptosis. Abrin is extremely lethal and considered as a potential agent for use in biological warfare. Currently, there are no antidotes or effective therapies available for abrin poisoning. Antibody based antitoxins function by either preventing toxin binding to cell surface receptors or by translocation. Antibodies against the B chain of RIPs function by inhibiting the binding of B chain of the toxin to cells, whereas the exact mechanism by which antibodies against A chain function is still not clear. The only known neutralizing monoclonal antibody against abrin A chain, namely, D6F10, was generated in our laboratory and was shown to rescue cells and mice from abrin intoxication. Earlier experiments with confocal microscopy suggested that mAb D6F10 could internalize in HeLa cells along with abrin, suggesting that the antibody can function intracellularly. Chapter 4 discusses the work carried out to delineate the mechanism of intracellular neutralization of abrin by the mAb D6F10. We observed significant reduction in binding and delay in abrin internalization in the presence of the neutralizing monoclonal antibody (mAb) D6F10. Considering that the majority of the abrin after internalization is removed by lysosomal degradation, we studied the fate of abrin in the presence of mAb D6F10. Confocal images did not show any difference in the distribution of abrin in the lysosomes in the absence or presence of antibody. However, the antibody remained persistently colocalized with abrin in the cells, suggesting that the antibody might inhibit enzymatic activity of abrin at its cellular site of action.

Epidemiological, experimental, and clinical observations provide support for a colorectal cancer chemopreventive role for aspirin. We have evaluated the effects of aspirin on proliferation biomarkers in normal-risk and high-risk human subjects for colorectal cancer. Colorectal biopsies were obtained at baseline and at 24h after 28 daily doses of 81mg of aspirin from 13 high-risk and 15 normal-risk subjects for colorectal cancer. We evaluated aspirin's effects on proliferating cell nuclear antigen (PCNA) immunohistochemistry and epithelial mucin histochemistry using the lectin, Amaranthus caudatus agglutinin (ACA) in crypt sections from rectal biopsies. The baseline whole crypt PCNA LIs differed significantly between normal-risk and high-risk subjects. PCNA LIs are not affected by 28 days of aspirin at 81mg daily. ACA LIs are decreased by 28 days of aspirin at 81mg daily in both normal-risk and high-risk subjects. Aspirin's effects on ACA LIs may have mechanistic and biological implications that deserve further attention. PCNA and ACA LIs are not useful as proliferation biomarkers for aspirin's chemopreventive activity in morphologically normal human colorectal mucosa.

No description available.