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Hereditary myopathy with lactic acidosis (HML, OMIM#255125) is an autosomal recessive disorder which originates from Västerbotten and Ångermanland in the Northern part of Sweden. HML is characterized by severe exercise intolerance which manifests with tachycardia, dyspnea, muscle pain, cramps, elevated lactate and pyruvate levels, weakness and myoglobinuria. The symptoms arise from malfunction of the energy metabolism in skeletal muscles with defects in several important enzymes involved in the TCA cycle and the electron transport chain. All affected proteins contain iron-sulfur (Fe-S) clusters, which led to the suggestion that the disease was caused by malfunctions in either the transportation, assembly or processing of Fe-S clusters. The aim of my thesis was to identify the disease causing gene of HML and to investigate the underlying disease-mechanisms. In paper I we identified a disease-critical region on chromosome 12; a region containing 16 genes. One of the genes coded for the Fe-S cluster assembly protein ISCU and an intronic base pair substitution (g.7044G>C) was identified in the last intron of this gene. The mutation gave rise to the insertion of intron sequence into the mRNA, leading to a protein containing 15 abberant amino acids and a premature stop. In paper II we investigated why a mutation in an evolutionary well conserved protein with a very important cellular role, which in addition is expressed in almost all tissues, gives rise to a muscle-restricted phenotype. Semi-quantitative RT-PCR analysis showed that the mutant transcript constituted almost 80% of total ISCU mRNA in muscle, while in both heart and liver the normal splice form was dominant. We could also show that, in mice, complete absence of Iscu protein was coupled with early embryonic death, further emphasizing the importance of the protein in all tissues. These data strongly suggested that tissue-specific splicing was the main mechanism responsible for the muscle-specific phenotype of HML. In paper III the splicing mechanisms that give rise to the mutant ISCU transcript was further investigated. We identified three proteins; PTBP1, IGF2BP1 and RBM39, that could bind to the region containing the mutation and could affect the splicing pattern of ISCU in an in vitro system. PTBP1 repressed the inclusion of the intronic sequence, while IGF2BP1 and RBM39 repressed the total ISCU mRNA level though the effect was more pronounced for the normal transcript. Moreover, IGF2BP1 and RBM39 were also able to reverse the effect of PTBP1. IGF2BP1, though not a splicing factor, had higher affinity for the mutant sequence. This suggested that the mutation enables IGF2BP1 binding, thereby preventing the PTBP1 induced repression seen in the normal case. In conclusion, we have determined the genetic cause of HML, identifying a base pair substitution in the last intron of the ISCU gene that gives rise to abnormally spliced transcript. The muscle-specific phenotype was also analyzed and tissue-specific splicing was identified as the main disease-mechanism. Furthermore, nuclear factors with ability to affect the splicing pattern of the mutant ISCU gene were identified. This work has thoroughly investigated the fundamental disease mechanisms, thus providing deeper understanding for this hereditary myopathy.

Orientador: José Moacir Marin / Banca: Sebastião Hetem / Banca: Patrícia Amoroso / Banca: Renata Camacho Miziara / Banca: Deny Munari Trevisani / Resumo: A aderência de bactérias patogênicas a receptores na superfície de . células epiteliais tem sido reconhecida como um importante evento inicial da colonização bacteriana. O principal mecanismo de patogenicidade da EPEC é uma lesão provocada pela sua aderência ao epitélio através do "attaching and effacing" (AlE), que é caracterizado pela íntima adesão da bactéria à célula do hospedeiro, esta adesão às microvilosidades do enterócito ocorre devido a formação de estruturas semelhantes a pedestais. O objetivo deste estudo foi desenvolver um modelo de infecção com a Escherichía calí enteropatogenica (EPEC) em camundongo do tipo mus musculus. A estes animais foram administrados um inoculo de 108 CFU de linhagens EPEC, sendo que a linhagem 3111-90 foi proveniente de cepas que podem causar diarréia infantil e as linhagens 537-1, 263, 304-3 e 988-2 foram provenientes de leite mastítico bovino. As linhagens foram introduzidas em camundongos com vinte dias de vida por via intraperitonial, e estes foram eutanasiados cinco horas após a inoculação. Como resultado através de cortes histológicos seriados, verificou-se que algumas das linhagens inoculadas levaram ao aparecimento de lesões suaves na mucosa intestinal, com a quebra da camada celular superficial da mucosa, aparecimento de células epiteliais com contornos irregulares e infiltração de células inflamatórias. / Abstract: The aim of this study was to develop a enteropathogenic Escherichia colí (EPEC) infection model in mice. An inoculum of 108 CFU of EPEC strains 3111-90 from infantile diarrhea; 537-1, 263,304-3 and 988-2 from bovine mastitic milk to newborn mice lead to a mildly damage in the intestinal mucosa with breaking in the superficial stratum, epithelial cells with irregular shape and a inflammatory cell infiltration. / Doutor

Preterm labour (PTL) is defined as labour before 37 completed weeks of gestation. Despite advances in medical research, PTL remains a major clinical problem. Preterm birth (PTB) rates range from approximately 5-18% worldwide. Importantly, PTB is the leading cause of childhood morbidity and mortality. PTL is difficult to predict and the aetiology is poorly understood but infection and inflammation are believed to be major factors. It has been suggested that the presence of intrauterine infection or inflammation may initiate the pathological, preterm activation of the inflammatory cascade associated with term labour. Therefore, PTL therapeutics should aim to inhibit these inflammatory pathways. Statins, 5-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, are potent inhibitors of cholesterol biosynthesis, which act on the mevalonate pathway. In addition to their lipid-lowering effects, statins also have anti-inflammatory and anti-contraction properties. The hypothesis of this thesis was that statins will prevent PTB by reducing inflammation. The aims of this thesis were firstly to investigate the effect of the statins, simvastatin and pravastatin, on inflammation and contractility in a pregnant human myometrial cell line. Secondly, to determine whether simvastatin and/or pravastatin can prevent PTB or improve neonatal outcome in a lipopolysaccharide (LPS)-induced mouse model of PTB. Myometrial cells were either co-treated with LPS and simvastatin/pravastatin, pretreated with simvastatin/pravastatin or treated with simvastatin/pravastatin post-LPS stimulation. The effect of statin treatment on the mRNA expression and the release of inflammatory mediators was then investigated. Simvastatin treatment reduced LPS-induced inflammation by both lowering the expression of pro-inflammatory mediators and increasing the expression of anti-inflammatory mediators. Pravastatin treatment did not alter the expression of inflammatory mediators following LPS stimulation. The effect of simvastatin on the contraction of myometrial cells was investigated by embedding the cells in rat tail collagen to form gels. As these are smooth muscle cells, basal contraction was observed causing the gel size to reduce. When LPS was introduced, this caused the gels to contract further than the vehicle treated gels. Simvastatin attenuated the contraction of the myometrial cells, both alone and in the presence of LPS. These effects were reversed by the addition of mevalonate pathway metabolites, mevalonate and geranylgeranyl pyrophosphate (GG-PP) but not by farnesyl pyrophosphate (F-PP). Simvastatin also lowered levels of phosphorylated myosin light chain (pMLC) in the myometrial cells, which is essential for smooth muscle contraction. Again, this effect was abolished by mevalonate and GG-PP but not F-PP. It is hypothesised that simvastatin attenuated myometrial cell contraction by inhibiting Rho isoprenylation by GG-PP, preventing Rho-associated kinase (ROCK) activation, which then prevented the phosphorylation of MLC. A mouse model of intrauterine LPS-induced PTB was utilised to investigate the effect of statin treatment on PTB and fetal survival. Mice received an intraperitoneal injection of pravastatin (10μg) or simvastatin (20μg or 40μg) on gestational day (D)16. This was followed by ultrasound-guided intrauterine injection of LPS (1μg) on D17 and another pravastatin/simvastatin treatment two hours later. When mice were treated with LPS, 77.8% of mice delivered preterm. When mice received LPS and 20μg simvastatin, 50% delivered preterm. However, when mice were treated with LPS and 40μg simvastatin, 40% delivered preterm, more pups were born alive and uterine pro-inflammatory mRNA expression was downregulated. Conversely, pravastatin did not prevent PTB or improve the percentage of live born pups. In summary, simvastatin treatment exerted anti-inflammatory and anti-contraction effects on human myometrial cells in vitro. The anti-contractile properties were likely due to the inhibition of the Rho/ROCK pathway. Furthermore, in our LPS-induced mouse model of PTB, fewer mice delivered preterm with simvastatin treatment, simvastatin attenuated LPS-induced pup mortality and reduced uterine inflammatory gene expression. These results suggest that statin therapy may be a novel treatment for PTL.

Eukaryotic Elongation Factor 1A (eEF1A) plays a key role in protein synthesis by delivering aminoacylated tRNAs to the A site of the ribosome. In higher vertebrates, two isoforms of eEF1A exist called eEF1A1 and eEF1A2, with eEF1A2 being expressed in adult brain, heart and skeletal muscle. Since 2012, several different de novo heterozygous missense mutations in EEF1A2 have been identified in humans and these cause epilepsy, intellectual disability and autism. Before considering treatment options, it is vital to determine whether these mutations cause loss or gain of protein function. I performed a battery of behavioural tests using two mouse lines with heterozygous loss of function mutations in eEF1A2. The aim was to determine whether there were any behavioural phenotypes consistent with intellectual disability and/or autism. Using heterozygous wasted mice (Eef1a2+/wst), I analysed the effects of aging on behaviour and found that Eef1a2+/wst mice showed reduced marble burying activity and reduced movement in the open field test with age. In a test of social behaviour, Eef1a2+/wst mice showed a significantly reduced preference for social novelty at all ages tested. The second heterozygous null line, Del22.ex3, was generated on a pure C57BL/6J genetic background. This new line was made in order to reduce the level of variation observed in data from the wasted line, which was on a mixed genetic background. The genetic background was shown to have an influence on behaviour as the results differed between this line and the wasted line. Del22.ex3 Eef1a2+/- mice showed significantly reduced engagement in repetitive behaviours compared with wild-type littermates and normal preference for social novelty. Using CRISPR/Cas9, a mouse line with the D252H missense mutation was generated and I repeated my behavioural testing on heterozygotes from this line. I found no behavioural abnormalities in this line suggesting a mouse-human difference in the ability to tolerate eEF1A2 missense mutations. Previous attempts to make a line with the G70S missense mutation were unsuccessful but as a product of this experiment, it was found that mice expressing G70S eEF1A2 had a comparable phenotype to and died at the same age as complete knockouts. This suggested that the G70S protein is non-functional and cannot compensate for loss of wild-type eEF1A2. These experiments have improved our understanding of the phenotypic effects of Eef1a2 mutations in mice and have shown, for the first time, that mutations in Eef1a2 affect mouse behaviour.

Obesity is associated with dysfunction of adipose tissue due to oxidative stress and inflammation, leading to insulin resistance. Thiosulfate sulfurtransferase (Tst) was previously identified as an adipose-expressed anti-diabetic gene that protects against diet-induced metabolic impairment when upregulated in adipose tissue of mice. TST is a mitochondrial enzyme involved in the metabolism of cyanide, reactive oxygen species (ROS) and endogenous hydrogen sulfide (H2S). This thesis tested the hypothesis that TST maintains metabolic health in the face of dietary obesity. To do this, I investigated the adipose-tissue phenotypes and metabolic consequences of Tst gene deletion (Tst–/– mice) and of adipose tissue-specific overexpression of human TST (Ad-hTST mice) after exposure to high fat diet (HFD). After 20 weeks of HFD, Tst–/– mice exhibited impaired glucose tolerance despite unchanged adipose tissue inflammatory cell infiltration, protein carbonylation and unfolded protein response activation. However, levels of mRNA encoding mitochondrial antioxidant enzymes including superoxide dismutase 2 and peroxiredoxin 3 were lower in Tst–/– mice on HFD. Unexpectedly, chow-fed Tst-/- mice had lower body weight and fat mass than wild-type controls highlighting a potential effect of Tst on fat accumulation with age. A new mouse model with high expression of human TST genetically targeted to adipose tissue (Ad-hTST) was developed using the LoxP / Cre recombinase expression system, with a parent line expressing Cre under the control of the adiponectin promoter to confer adipose specificity. The Ad-hTST mice were found to gain a similar amount of weight and fat mass to control mice when exposed to 6 weeks of HFD. However, Ad-hTST mice had impaired glucose tolerance with no change in inflammatory cell infiltration, mRNA levels of antioxidant enzymes or unfolded protein response genes. Thus, unexpectedly, overexpression of human TST in adipose tissue of mice results in a detrimental metabolic phenotype. In vivo and in vitro experiments were conducted to test the hypothesis that TST protects against ROS accumulation. Paraquat was tested as an inducer of oxidative stress in vivo in wild-type, Tst-/- and Tst+/- mice. At the doses used (25mg/kg and under), mice became unwell and lost weight, with no increase in markers of oxidative stress in adipose or lung. The production of mitochondrial ROS in response to exogenous hydrogen peroxide (H2O2) exposure was increased in primary adipocytes from Tst-/- mice in vitro. However, primary hepatocytes showed reduced mitochondrial ROS production in response to H2O2 exposure. ROS production in hepatocytes was unaffected by pre-incubation with a H2S donor, an inhibitor of H2S-producing enzyme CSE or N-acetyl-cysteine, an antioxidant. TST may therefore influence mitochondrial ROS production differently in cell types such as adipocytes and hepatocytes. Disposal of exogenous H2O2 was unchanged in primary adipocytes from Tst-/- and Ad-hTST mice, and this was not affected by pre-incubation with sodium thiosulfate, a TST substrate. Metabolic changes in response to HFD may be influenced by alteration in TST expression, however the current data suggest it is unlikely to occur through the prevention of excessive local ROS accumulation in adipose tissue. Mice lacking the Tst gene globally and mice with adipose-specific overexpression of the human TST gene have a similarly impaired metabolic response to HFD. The phenotype of adipose-specific human TST-overexpressing mice does not recapitulate the protective metabolic phenotype produced by overexpression of the endogenous mouse Tst gene. In conclusion, TST may influence adipose tissue due to its role in the oxidation of H2S, however, by the current means, it does not appear to substantially impact the response of this tissue to oxidative stress.

Introduction: Hepatic stellate cells (HSC) and myofibroblasts may be relevant stromal drivers of human hepatocellular carcinoma (HCC). It was hypothesised that targeted inhibition of αv integrin-mediated TGF-β activation, by HSC or hepatocytes, may result in reduced peri-tumoural and intra-tumoural extracellular matrix formation, and reduced hepatic carcinogenesis. The role of HSC in acute liver injury is less well characterised. It was anticipated that integrin signalling on HSC and hepatocytes might also be relevant in the acute setting. The emerging technique of intravital microscopy (IVM) allows detailed, real-time investigation of the cellular processes involved in hepatocyte injury, cell death and repair. It was hypothesised that this could be coupled with mouse models of HCC and acute liver injury, to perform sequential imaging under anaesthesia. Aims: (i) To determine the effect of targeted inhibition of αv integrins on HSC and hepatocytes, during hepatocarcinogenesis, in a mouse model of HCC. (ii) To investigate the effect of targeted inhibition of αv and other integrins on HSC, hepatocytes, and liver sinusoidal endothelial cells (LSEC), during acute liver injury, in the mouse model of paracetamol-induced liver injury. (iii) To develop IVM of the liver, via an abdominal imaging window, with optimisation of surgical and imaging techniques, to allow sequential imaging of the same animal. Methods: The diethylnitrosamine (DEN)-induced mouse model of hepatocarcinogenesis was used, and PDGFRβ-Cre;αvfl/fl and Alb-Cre;αvfl/fl mice were employed to deplete αv integrins on HSC and hepatocytes respectively. Tumours were harvested at 40 weeks post-DEN. Tumour size and number was evaluated in all animals. PDGFRβ-Cre;αvfl/fl and Alb-Cre;αvfl/fl mice were used in the paracetamol model, to investigate the role of αv integrins in acute liver injury. PDGFRβ-Cre;β8fl/fl and Alb-Cre;β 8fl/fl animals were also tested in this model. The role of integrins in liver sinusoidal endothelial cells (LSEC) during paracetamol-induced liver injury was evaluated using Cdh5-Cre mice. IVM of the liver was performed by surgical implantation of an abdominal imaging window, consisting of a titanium ring and coverslip, secured in place with a purse string suture. Fluorescent reporter mice were used to identify hepatic and vascular architecture, and other label-free microscope technologies were utilised to image collagen, lipid distribution, necrotic areas and blood flow within tissues. Results: In large cohorts of PDGFRβ-Cre;αvfl/fl, Alb-Cre;αvfl/fl, and control animals, there was no difference in mean tumour size or number, at 40 weeks. Targeted inhibition of α v integrins and β 8 integrin on hepatocytes, HSC or LSEC was not protective in paracetamol-induced liver injury. IVM of the liver can be performed on animals with HCC and throughout paracetamol-induced liver injury, to obtain high quality, real-time images of multiple cell lineages and the hepatic microenvironment. Conclusions: The role of TGF-β in HCC pathogenesis is complex and context-dependent. Targeted loss of αv integrin did not result in reduction in tumour burden in this non-cirrhotic model of HCC. IVM of the liver is a powerful tool to quantify inflammatory infiltrates and assessment of vascular remodelling throughout the course of acute liver injury and regeneration, providing insights into the biological processes determining recovery.

Androgens are well documented as important regulators of male health, primarily in the maintenance and development of male sexual characteristics. However, a decline in circulating androgens has also been associated with co-morbidities such as obesity, cardiac disease and metabolic syndrome. Previous research has focussed upon the body wide impact of adrenal androgens, however whilst androgen receptor (AR) is abundantly expressed in the adrenal cortex of both rodents and humans, surprisingly little is known about androgen action on the adrenal cortex itself. This gap in our understanding is at least in part due to the perceived lack of suitable animal models. Rodents have largely been overlooked as a model system as their adrenals are unable to produce androgens due to lack of 17α Hydroxylase and 17, 20 lyse activity and they therefore do not have a zona reticularis. However, historical studies using castrated mice showed that removal of androgens leads to the redevelopment of an additional cortex zone known as the transient X-zone. The foetal adrenal is thought to give rise the adult adrenal cortex in human and rodents. These foetal cells are maintained for a period postnatally and regress differently depending on species and sex. In the human this zone is known as the ‘foetal zone’, and the rodent homologue termed the ‘X-zone’. The mechanisms underpinning the regression of the X-zone and its purpose and maintenance postnatally still aren’t clearly understood. To provide a comprehensive overview of androgen signalling in the adrenal cortex, multiple mouse models were utilised. First, Cre/loxP technology was used to ablate AR specifically from the adrenal cortex. Further androgen manipulation was achieved through castration (removal of androgens) and human chorionic gonadotropin (hCG) treatment (increased androgens). The initial study investigates the impacts on the male mouse adrenal. Histology analysis revealed the presence of an X-zone in all experimental cohorts following loss of AR or circulating androgens, confirmed by 20- α-hydroxysteroid dehydrogenase (20 alpha-HSD) expression. These data demonstrate that androgens signalling via AR is required for X-zone regression during puberty. However, interrogation of morphology of hCG treated cohorts revealed no phenotypic changes compared to controls, this demonstrates that hyper stimulation with androgens does not negatively impact the adrenal cortex or influence X-zone morphology. Differences in X-zone morphology and 20 alpha-HSD localization prompted cortex measurements which revealed significant differences in X-zone depth and cell density depending on ablation of AR, circulating androgens or both. This suggests that androgens and androgen receptor are working together and also independently to regulate the adrenal cortex. This result was strengthened through analysis of steroid enzyme genes and cortex markers, which revealed that normal AKR1B7 expression was absent following loss of androgens but not androgen receptor. A final part of this study examined the impacts long term androgen receptor ablation and long term castration in ageing animals. A final part of this study examined the impacts long term androgen receptor ablation and long term castration in ageing animals. These results demonstrate that following prolonged loss of androgens that there is no major disruption to the adrenal cortex. Morphology analysis and X-zone measurements revealed that X-zone regression was occurring in mice with long term castration, characterized by a reduction in size and pockets of vacuolization throughout the X-zone. This phenotype is also observed in ageing females with X-zone regression via vacuolization. These data suggest that following prolonged loss of androgens, the male adrenal is feminized and behaves as such. In contrast, AR ablation only, results in an enlarged adrenal with large spindle cell lesions and X-zone expansion confirmed by X-zone measurements. Initial experiments have demonstrated that androgens can work independently of AR to regulate the adrenal cortex. Together these data suggests that AR is required to control the appropriate action of circulating androgens in the adrenal cortex, with loss of AR resulting in off target signalling from circulating androgens in the adrenal leading to spindle cell hyperplasia, X-zone expansion and X-zone mislocation. A second set of studies were carried out to determine the role of androgen signalling in the female adrenal, specifically, if loss of AR leads to the absence of normal X-zone regression during pregnancy. To answer this question the same selective AR ablation model was used. Analysis of litters comparing observed and expected genetic distribution revealed significantly fewer females being born carrying complete ablation of adrenal AR. Morphology analysis of these mice revealed severe cortex disruption and spindle cell hyperplasia similar to that observed in mutant males. Investigation of adrenals following pregnancy revealed that X-zone regression still occurred despite loss of AR. This result shows that X-zone regression in the female is under different regulation compared to male adrenal and occurs via an androgen-independent signalling mechanism. However, loss of AR still leads to anatomical dysregulation of the adrenal cortex. AR ablation revealed changes in glucocorticoid receptor (GR) expression in the adrenal cortex. To dissect this relationship further a final study was conducted, attempting to ablate GR from the adrenal cortex also using the Cyp11a1 Cre. Initial observations of these mice revealed excessive hair loss through barbering, curved spines and stressed behaviour when monitored in the cage under normal conditions. Immunohistochemistry was used to confirm GR ablation in the adrenal cortex, however, to our surprise, GR expressing cells were not steroidogenic and thus were not targeted by the Cre recombinase. Despite no GR ablation in the adrenal, morphology analysis revealed severe disruption to the adrenal cortex. The Cyp11a1 Cre not only targets the adrenal but is expressed in the hindbrain. To determine if GR ablation in the hindbrain explains the phenotype, we next used PCR analysis interrogating hindbrain genomic DNA to determine if there was recombination of GR. Results confirmed GR recombination in the hindbrain. Due to the observation of stressed behaviour and adrenal cortex disruption, we wanted to determine if this was a result of hyperactivity of the adrenal cortex. Serum corticosterone was analysed and was elevated in these animals. These data revealed that GR ablation in the hindbrain results in adrenal cortex disruption and an elevated stress response, potentially highlighting a new model to investigate stress disorders and their impact on the hypothalamic-pituitary-adrenal axis. Together this data defines new roles for AR signalling in the adrenal cortex and the role of the hindbrain GR signalling in regulating adrenal morphology and function.

abstract: Breast cancer is the most common cancer and currently the second leading cause of death among women in the United States. Patients’ five-year relative survival rate decreases from 99% to 25% when breast cancer is diagnosed late. Immune checkpoint blockage has shown to be a promising therapy to improve patients’ outcome in many other cancers. However, due to the lack of early diagnosis, the treatment is normally given in the later stages. An early diagnosis system for breast cancer could potentially revolutionize current treatment strategies, improve patients’ outcomes and even eradicate the disease. The current breast cancer diagnostic methods cannot meet this demand. A simple, effective, noninvasive and inexpensive early diagnostic technology is needed. Immunosignature technology leverages the power of the immune system to find cancer early. Antibodies targeting tumor antigens in the blood are probed on a high-throughput random peptide array and generate a specific binding pattern called the immunosignature. In this dissertation, I propose a scenario for using immunosignature technology to detect breast cancer early and to implement an early treatment strategy by using the PD-L1 immune checkpoint inhibitor. I develop a methodology to describe the early diagnosis and treatment of breast cancer in a FVB/N neuN breast cancer mouse model. By comparing FVB/N neuN transgenic mice and age-matched wild type controls, I have found and validated specific immunosignatures at multiple time points before tumors are palpable. Immunosignatures change along with tumor development. Using a late-stage immunosignature to predict early samples, or vice versa, cannot achieve high prediction performance. By using the immunosignature of early breast cancer, I show that at the time of diagnosis, early treatment with the checkpoint blockade, anti-PD-L1, inhibits tumor growth in FVB/N neuN transgenic mouse model. The mRNA analysis of the PD-L1 level in mice mammary glands suggests that it is more effective to have treatment early. Novel discoveries are changing understanding of breast cancer and improving strategies in clinical treatment. Researchers and healthcare professionals are actively working in the early diagnosis and early treatment fields. This dissertation provides a step along the road for better diagnosis and treatment of breast cancer. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2015