Objective: Treatment compliance in children with Crohn’s disease is associated with higher levels of symptom remission. We hypothesized that the management, comorbidities, and complications for children with Crohn’s disease would differ based on a diagnosis of noncompliance. Methods: Using the Kids’ Inpatient Database for 2006-2012, we identified young patients (<21 >years) with a diagnosis of Crohn’s disease. Diagnoses and procedures were analyzed according to a recorded diagnosis of noncompliance. Multivariable logistic regression analysis was performed to examine the association between noncompliance and the outcomes of interest. Results: There were 28,337 pediatric Crohn’s disease hospitalizations identified with 1,028 (3.6%) hospitalizations having a diagnosis of both Crohn’s disease and noncompliance. The mean age of the study population was 15.9 years and 48.9% were girls. Black patients ( multivariable adjusted odds ratio, aOR,2.27; 95% CI:1.84-2.79) and those in the lowest income quartile (aOR 1.57; 95% CI:1.20-2.05) had an increased likelihood of a noncompliance diagnosis than respective comparison groups. Noncompliant patients had an increased likelihood of concurrent depression, nutritional deficiency, and anemia. Patients with a diagnosis of noncompliance had lower rates of intestinal obstruction (4.0% vs 6.3%), intraabdominal abscesses (2.0% vs 4.2%,), and underwent fewer major surgical procedures (aOR 0.40; 95% CI:0.31-0.53) and large bowel resections (aOR 0.44; 95% CI:0.31-0.64) than patients without this diagnosis. Conclusions: We found significant differences in socioeconomic status and race among hospitalized children with Crohn’s disease with, as compared to those without, a diagnosis of noncompliance. Children with noncompliance have different comorbidities, disease-related complications, and are managed differently. Possible explanations for observed treatment differences include a reluctance to offer surgery to those with a diagnosis of noncompliance, a refusal of intervention by noncompliant patients, or implicit bias. Further investigation is warranted to better define noncompliance in this population and to determine the implications of this diagnosis.

Seminal fluid proteins (SFPs) have been shown to affect the physiology,behaviour and immune responses of mated females in some species. Thisopen window for manipulation of female’s fitness allows the possibility forcomplex evolutionary dynamics between the SFPs and proteins of femalesthat would counter the effects of the former, the female reproductive proteins (FRPs). Also, the bean beetles of the Bruchinae subfamily are pests to pre-ferred species of plant hosts. The hosts have a great variety of secondary defensive metabolites between them and to detoxify those compounds, each beetle species is expected to have a well adapted arsenal of digestive proteinsfor a specific host. I carried out a comparative study with four species of bean beetles with the aim to identify patterns of selection in the proteins mentioned. Expression data for one of those species, Callosobruchus maculatus, has allowed to identify its SFPs, FRPs and digestive proteins and with orthology inference I identified their orthologues in the other three species. Then I estimated theratio of non-synonymous to synonymous substitution rates (ω) for each protein by using codeML of the PAML package and used them as a proxy for estimating selection. FRPs had about the same ω values as conserved genes found across the Arthropod phylum, while the SFPs and digestive proteins hadhigher ω values, indicating more relaxed purifying selection. I also performed tests of positive selection and have identified 92 digestive proteins, 9 FRPs and 26 SFPs as potential targets for future functional work. Finally, I examined the scenario of co-evolution between SFPs and FRPs because of direct interaction. By correlating branch-specific ω values for each possible pairs of proteins I found that SFPs are associated on average more with FRPs than with digestive or conserved genes, as expected. The same was true for the FRPs. Also I examined the possibility of factors contributing to the association such as expression levels, sex-biased expression and protein function. Using linear regression models I found that expression levels and proteinfunction do predict in some degree the ω estimates and could thus also affectthe correlations examined. High gene expression levels reduce the overall ωvalues of genes, also known as E-R anticorrelation. Sex-biased expression does not affect the overall ω values, but does affect the intensity of the E-R anticorrelation, with it being less prominent in male-biased genes and more prominent towards female-biased genes.

Nonalcoholic steatohepatitis (NASH) is a severe liver disorder characterized by triglyceride accumulation, severe inflammation, and fibrosis. With the recent increase in prevalence, NASH is now the leading cause of liver transplantation, with no approved therapeutics available. Despite years of research, the exact molecular mechanism of NASH progression is not well understood, but fat accumulation is believed to be the primary driver of the disease. Therefore, diacylglycerol O-acyltransferase 2 (DGAT2), a key enzyme in triglyceride synthesis, has been explored as a NASH target. RNAi-based therapeutics is revolutionizing the treatment of liver diseases, with recent chemical advances supporting long term gene silencing with single subcutaneous administration. Here we identified a hyper-functional, fully chemically stabilized GalNAc conjugated siRNA targeting DGAT2 (Dgat2-1473) that upon injection elicits up to three months of DGAT2 silencing (>80-90%, p<0.0001) in wild-type and NSG-PiZ “humanized” mice. Using an obesity-driven mouse model of NASH (ob/ob-GAN), Dgat2-1473 administration prevents and reverses triglyceride accumulation (> 50%, p:0.0008), resulting in significant improvement of the fatty liver phenotype. However, surprisingly, the reduction in liver fat didn’t translate into a similar impact on inflammation and fibrosis. Thus, while Dgat2-1473 is a practical, long-lasting silencing agent for potential therapeutic attenuation of liver steatosis, combinatorial targeting of a second pathway may be necessary for therapeutic efficacy against NASH.

Osteopetrosis is a metabolic bone disease in mammals characterized by a generalized skeletal sclerosis caused by reduced bone resorption. This reduced bone resorption is manifested in afflicted animals by abnormal bone shape, reduced or absent marrow cavities, extramedullary hemopoiesis, abnormal mineral homeostasis and absent or delayed tooth eruption. The available osteopetrotic animal mutations have been a constant source of fruitful investigations concerning the systemic regulation of osteoclastogenesis and bone metabolism. Tooth eruption, on the other hand, is a localized manifestation of the timely activation of bone resorption and bone formation on opposite sides of an erupting tooth. Its rate-limiting step is the speed of bone resorption to form the eruption pathway. In this dissertation, we used two osteopetrotic rat mutations, toothless (tl) and microphthalmia blanc (mib), to investigate the abnormal development of osteoclasts and tooth eruption in mutant rats with an emphasis on the role of systemic and local factors. The significant contributions to this work are listed below. 1. In the toothless rat, a mutation lacking erupted dentition due to severely reduced bone resorption, colony-stimulating factor-1 (CSF-1) promoted tooth eruption but this was delayed compared to normal rats. Eruption was accompanied by changes in the populations of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear cells in the dental follicle and TRAP+ osteoclasts on adjacent alveolar bone surfaces. These cell populations were dramatically increased in treated mutants compared to untreated tl rats, but the timing of their appearance was delayed compared to normal littermates. This lag in the appearance of osteoclasts and their precursors corresponded to the delay in eruption of first molars in treated tl rats. 2. CSF-1 also accelerated the eruption of molars in normal rats. CSF-1 increased the number of TRAP+ mononuclear cells in the dental follicle and TRAP+ osteoclasts on adjacent alveolar bone surfaces, but had no effect on the timing of their appearance in normal rats. 3. Our data revealed a differential effect on tooth eruption of the growth factors CSF-1 and epidermal growth factor (EGF). CSF-1 accelerated eruption of molars in normal rats, but had no effect on incisor eruption. On the other hand, EGF accelerated incisor eruption; but did not affect molar eruption in normal rats. 4. We have described the mechanism for the transient, mild form of osteopetrosis inherited by mib rats. Mutant animals possess a typical sclerosis at birth, which diminished--but was not resolved--during the first postnatal month. These characteristics are caused by early reductions in osteoclast number and function which improve to normal levels by 4 weeks. Osteoclast numbers were severely reduced in mib rats between birth and 2 weeks, but improved to near normal levels by 4 weeks. Neonatal abnormalities in osteoclast function included reduced staining for the functional enzymes TRAP and TrATPase, decreased levels of mRNA for both TrATPase and CAll, and inability to form a well-developed ruffled border. None of these defects were apparent after the first postnatal month. 5. Finally, we have shown that the dental abnormalities caused by the mild, transient form of osteopetrosis in mib rats are limited to incisor defects and delayed eruption of all teeth. Histologic and radiographic examination of mutant incisors revealed that, contrary to the situation in normal rats, the apex of the incisors of mib rats failed to extend past the first molar region to the third molar. The incisor apex of newborn mib rats was misshaped due to ankylosis of incisor matrices with alveolar bone. This ankylosis was temporary, being resolved by the third postnatal day. The delayed eruption of incisors in mib rats and abnormal shape and occlusion of these teeth in older animals is a consequence of the temporary ankylosis in newborn rats.

L'ADN tumoral circulant (ADNtumc) est apparu depuis plusieurs années comme un biomarqueur prometteur susceptible d'apporter des informations permettant l'optimisation de la prise en charge du patient en oncologie. L'objectif de cette thèse était double et s'articule autour de deux axes : i) évaluer différentes conditions préanalytiques et analytiques (digitale PCR (dPCR) principalement) pour la détection de ce biomarqueur ii) évaluer l'intérêt clinique potentiel de ce biomarqueur en oncologie digestive. La première partie rapporte 3 travaux (3 articles originaux dont une collaboration nationale (équipe parisienne dirigée par J. Tost)). Dans le travail n°1, nous avons montré la faisabilité de détecter l'ADNtumc par dPCR directement à partir du plasma de 43 prélèvements de patients avec cancer colorectal métastatique (CCRm). Il n'y avait pas de différence significative pour le taux de détection des mutations KRAS circulantes entre les groupes avec et sans extraction d'ADN (93 % (40/43) versus 88 °A) (38/43), respectivement). Dans le travail n°2, nous avons mis au point une méthode basée sur l'apport d'héparinase pour la détection d'ADNtumc à partir de 194 prélèvements héparinés de patients suivis en oncologie. Ce traitement des échantillons par l'héparinase permettait l'analyse de l'ADNtumc pour 117/194 (60 %) patients avec inhibition Préalable de la dPCR par l'héparine. Enfin, dans le travail n°3, nous avons comparé plusieurs plate-formes de détection d'ADNtumc et montré que la dPCR affichait des résultats de détection comparables sur le plan qualitatif et quantitatif avec une plateforme ultrasensible d'Enhanced-ice-COLD-PCR (E-ice-COLD-PCR) pour les échantillons avec une fréquence allélique d'ADNtumc >0,4 °A La deuxième partie rapporte 3 travaux (3 articles originaux) sur l'intérêt clinique de la détection d'ADNtumc par dPCR en oncologie digestive. Nous avons ainsi montré que ce biomarqueur conférait un intérêt diagnostique (travail n°4), Pronostique (travail n 4 à 6) et prédictif de la réponse aux traitements (travail n°6) chez les Patients avec adénocarcinome pancréatique (AP) (travail n°4) et CCRm (travail n°5 à 6). / For several years, circulating tumor DNA (ctDNA) has emerged as a promising biomarker providing relevant information to optimize patient care in oncology. The aim of this thesis was both: (i) to evaluate different preanalytical and analytical conditions (digital PCR (dPCR) mainly) for the detection of this biomarker; (ii) to evaluate the potential clinical interest of this biomarker in digestive oncology. The first part reports 3 works (3 original articles including a national collaboration (Parisian team led by J. lost)). In work no. 1, we have shown the feasibility of ctDNA detection by dPCR directly from the plasma of 43 samples from patients with metastatic colorectal cancer (mCRC). There was no significant difference in the detection rate of circulating KRAS mutations between groups with and without DNA extraction (93% (40/43) versus 88% (38/43), respectively). In work no. 2, we developed a method based on the heparinase addition for the ctDNA detection from 194 heparinized samples of patients followed in oncology. This treatment of samples by heparinase allowed the ctDNA analysis of 117/194 (60%) patients with prior inhibition of dPCR by heparin. Finally, in work no. 3, we compared several ctDNA detection platforms and snowed that dPCR displayed qualitatively and quantitatively comparable detection results with an ultrasensitive platform of E-ice-COLD-PCR for the samples with ctDNA allelic fraction ?.0 4%. The second part reports 3 works (3 original articles) on the clinical interest of the ctDNA detection by dPCR in digestive oncology. We have thus shown that this biomarker had a diagnostic (work no. 4). prognostic (works no. 4 to 6) and predictive response to treatments (work no. 6) interest in patients with pancreatic adenocarcinoma (work no. 4) and mCRC (works no. 5 to 6).

This study describes, on a comparative basis, the morphology of mechanical (the linguo-laryngeal apparatus) and sensory (Herbst corpuscles and taste buds) specialisations in the upper digestive tract (bill and oropharynx) of the ostrich and emu, with a view to a better understanding of the functional significance of these structures. The ostrich and emu are commercial entities that constitute important niche industries and are farmed intensively throughout South Africa. A lack of information on the mechanical and sensory specialisations of the upper digestive tract in these two birds hampers a sound understanding of food selection and intake. A total of 48 adult (12-14 months) ostrich heads and 48 adult emu (12-14 months) heads obtained from birds at slaughter at commercial abattoirs and farms, as well as 5 ostrich chick (2-4 weeks) heads and 1 emu chick (8 weeks) head, obtained from previous research projects, were used for this study. Morphological features were described using basic gross anatomical (dissection and stereomicroscopy) and histological techniques (H&E staining), supplemented by differential staining for cartilage and bone, transmission electron microscopy and immunohistochemistry. The findings of the study were compared with the relevant literature and hypotheses for functional significance were formed. The avian glottis channels air from the oropharynx to the trachea and is situated on an elevated structure, the laryngeal mound. It is imperative that the glottis be protected and closed during swallowing, which in mammals is achieved by covering the glottis with the epiglottis, as well as by adduction of the arytenoid cartilages. An epiglottis, however, is reportedly absent in birds. Ratites such as the ostrich and emu possess a very wide glottis in comparison to other birds. The question therefore arises as to how these large birds avoid inhalation of food particles through a wide glottis, with apparently little protection, particularly as their feeding method involves throwing the food over the glottis to land in the proximal esophagus. In the ostrich, when the glottis was closed and the tongue body retracted, the smooth tongue root became highly folded and the rostral portion of the laryngeal mound was encased by the pocket in the base of the ∩-shaped tongue body. In this position the lingual papillae also hooked over the most rostral laryngeal projections. However, in the emu, retraction of the tongue body over the closed glottis resulted in the prominent, triangular tongue root sliding over the rostral portion of the laryngeal mound. In both the ostrich and emu these actions resulted in the rostral portion of the laryngeal mound and weakest point of the adducted glottis being enclosed and stabilised. Only after conducting a comparative study between these two birds using fresh specimens did it become clear how specific morphological peculiarities were perfectly specialised to assist in the closure and protection of the wide glottis. A unique anatomical mechanism in ratites was identified, described and proposed, which may functionally replace an epiglottis; the linguo-laryngeal apparatus. The oropharynx of the ostrich and emu is richly supplied with Herbst corpuscles. This widespread distribution of these mechanoreceptors has not previously been reported in birds. Specific concentrations of Herbst corpuscles within the oropharynx, which differ between the ostrich and emu, assist in the accurate positioning of the tongue and laryngeal mound for cleaning the choana (internal nares). The Herbst corpuscles are strategically located to aid in the handling and transport of food and the median palatine and ventral ridges in the ostrich display a concentration of Herbst corpuscles which denote these structures as sensory organs, namely the palatal and interramal organs. Three specific arrangements of Herbst corpuscles were noted in the oropharynx. The first arrangement consisted of groups of corpuscles located peripherally around a myelinated nerve and was present in the bill tip. The second arrangement, possibly linked to the first, was that of individual or groups of corpuscles without an obvious associated nerve and was present throughout the remaining regions of the oropharynx. The third arrangement was that of corpuscles associated with large, simple branched tubular mucus-secreting glands. The basic structure of Herbst corpuscles in the ostrich and emu, observed by light and transmission electron microscopy, of a capsule (with cellular and acellular lamella), an outer zone (collagen fibrils, fibroblasts and a fluid matrix), an inner core (formed by bilaterally symmetrical specialised Schwann cells) and a receptor axon, is similar to that noted for other avian species. However, unlike in other birds, the capsule of the Herbst corpuscle in the ostrich and emu is formed by myofibroblasts which indicates contractile properties for this component of the corpuscle in ratites. Sensory cilia were noted in the myofibroblasts of the capsule and fibroblasts of the outer zone of the ostrich Herbst corpuscle which may assist in regulating the tension of the capsule. These features have not been reported in other avian species. Although the structure of the palaeognathous palate has been widely studied, relatively little information is available on the morphology of the ratite bill. The kiwi possesses a bill tip organ and the present study confirmed the existence of this somatosensory organ in the ostrich and emu. Examination of the rhamphotheca of these two birds demonstrated numerous specialisations. In the emu, rhamphothecal serrations with intervening keratinised pegs on the rostral mandibular tomia resembled a form of pseudo-teeth. These structures may share a similar embryological origin to teeth; however, they would appear to function by channelling and enhancing vibratory stimuli to Herbst corpuscles in nearby bony pits. In the ostrich, epidermal troughs were present in the regions overlying the bill tip organ and functioned to enhance vibratory stimuli to the underlying Herbst corpuscles. Additionally, in the ostrich only, and not related to the structure or functioning of the bill tip organ, the rostral tomia and maxillary and mandibular nails were composed of typical tubular and inter-tubular horn. This may represent a unique feature in birds. The structure of the mandible and premaxilla was similar to that described previously for these birds. However, the persistence of Meckel’s cartilage through to the adult bird in the ostrich and emu is a novel avian feature not previously reported. The bony bill tips were adorned with numerous sensory (bony) pits which displayed similar distribution patterns in the ostrich and emu and indicated the presence, macroscopically, of a bill tip organ. The total number of pits in the bill tip of the ostrich and emu did not differ significantly, although regional differences did occur. The sub-divisions of the trigeminal nerve (N. opthalmicus R. medialis and N. intramandibularis) innervating the bill tip were well developed in both birds and displayed extensive branching. The emu displayed more myelinated nerve fibres in both nerves than in the ostrich. As myelinated nerve fibres supply Herbst corpuscles, the number of nerve fibres is correlated to the number of corpuscles. No correlation could be made between the number of pits in a particular region and the number of nerve fibres or with the relative percentage of Herbst corpuscles in that region. The bill tip organ in both species was basically similar except for the epidermal specialisations noted above. Two parts of the bill tip organ were recognised; the bony bill tip organ (Herbst corpuscles stacked in bony cavities and pits) and the peripheral bill tip organ (Herbst corpuscles in sheets or chains in the connective tissue between the epithelium and bone). The morphology of the bill tip organ in the ostrich and emu indicates that it is an organ that functions by direct touch. These two ratite species appear to possess the most elaborate bill tip organ of any pecking bird. The existence of a bill tip organ in the ostrich and emu is an enigma and points to the possibility that a bill tip organ is a basal structure in all palaeognathous birds (living and extinct). Furthermore, it is evident by observing the exploratory behaviour of the ostrich and emu, that they use their bill tip organ extensively as a tool for exploring and interpreting their environment as well as for discriminating food. The sense of taste in birds is an important motivator for feeding as well as initial food selection. The existence of this sense in ratites has remained largely speculative. In the present study taste buds were only identified in the emu and were predominantly located in the caudal region of the non-pigmented oropharyngeal roof and sparsely located on the oropharyngeal floor. The taste buds extended the full width of the epithelium in which they were located and were ovoid structures. The taste bud was composed of centrally located, vertically oriented light and dark cells (representing both receptor cells and supporting elements) and peripherally situated follicular cells which were continuous with the surrounding Str. germinativum of the stratified squamous epithelium. Positive IHC labelling for neurofilament demonstrated numerous fine nerve fibres (Neurofibra gustatoria) within the connective tissue immediately surrounding the taste bud. Taste bud morphology in the emu was similar to that described in other birds. However, when sectioned tangentially they were indistinguishable from the surrounding epithelium with H&E staining. By using IHC labelling, concentrations of nerve fibres could be demonstrated beneath apparently nondescript epidermal structures, thus indicating the presence of a taste bud. The distribution of taste buds in the oropharynx could be linked to the particular feeding method of the emu. Based on information from GenBank, it would appear that the relatively few taste buds present in the emu oropharynx would mainly function in distinguishing bitter taste. As bitter-tasting compounds can cause a negative association with a particular food type, it would appear that the sense of taste in the emu would predominantly function for protection and not food selection. This study revealed various unique findings regarding the mechanical and sensory specialisations in the upper digestive tract of the ostrich and emu.  The ostrich and emu possess a combination of structures which functionally replace an epiglottis, namely the linguo-laryngeal apparatus.  Herbst corpuscles are widely distributed in the oropharynx of the ostrich and emu and their distribution is related to the particular feeding habits of these birds.  The capsule of Herbst corpuscles in the ostrich and emu is composed of contractile elements, a feature not reported in other birds.  The ostrich and emu possess a well-developed bill tip organ, which is an unusual feature amongst pecking birds.  Taste buds are present in the emu and no structures resembling taste buds were identified in the ostrich. / Thesis (PhD)--University of Pretoria, 2013. / gm2014 / Anatomy and Physiology / Unrestricted

The pancreas is located below the liver and adjacent to the small intestine where it connects to the duodenum. It consists of exocrine and endocrine components. The exocrine portion makes enzymes which are deposited in the duodenum to digest fats, proteins, and carbohydrates. Exocrine tissue also makes bicarbonates that neutralize stomach acids. The endocrine portion produces hormones such as insulin and glucagon which are released into the blood stream. These hormones regulate glucose transport into the body's cells and are crucial for energy production. The pancreas is associated with diseases such as cancer, diabetes, Annular pancreas and Nesidioblastosis. Annular pancreas and Nesidioblastosis are congenital malformations associated with excess endocrine tissue of the pancreas and its structures. Understanding the development of the pancreas might lead to insight of these diseases. The pancreas arises from the endoderm. In zebrafish, Nodal signaling activates mix-type and gata genes that then function together to regulate sox32 expression which is necessary and sufficient to induce endoderm formation. Interestingly, sox32 is exclusive to zebrafish and works synergistically with pou5f1 to regulate its own expression and turn on sox17 expression. sox17is evolutionarily conserved from zebrafish to mouse and is necessary for endoderm formation. Signals from within the endoderm and the surrounding mesoderm specify regions in the endoderm to develop into the pancreas and other endodermal organs. Sonic hedgehog (shh) expression in the foregut establishes the anterior boundary of the pancreas primordium while cdx4 expression establishes the posterior boundary, but what regulates these factors is unclear. We determined that two Three Amino Acid Loop Extension (TALE) homeodomain transcription cofactors, Meis3 and Pbx4, regulate shh expression in the anterior endoderm. Disrupting either meis3 or pbx4 reduces shh expression in the anterior endoderm. As a result, anterior ectopic insulin expression occurs outside the normal pancreatic domain. Therefore, we discovered upstream regulatory factors of shhexpression in the anterior endoderm, which is necessary for patterning the endoderm and pancreas primordium. We performed an ENU (N-ethyl-N-nitrosurea) haploid screen to look for endocrine pancreas mutants and to find other factors involved in pancreas development and patterning. From the screen, we characterized two mutants. We identified an aldh1a2 mutant, aldh1a2um22, which blocks the production of Retinoic Acid (RA) from vitamin A. While RA is known to be necessary for differentiation of the pancreas and liver, we also found it to be necessary for intestine differentiation. Two other aldh family genes exist in the zebrafish genome, but our data suggests that aldh1a2is the only Aldh that functions in endoderm differentiation and it is maternally deposited. From the screen, we discovered a second mutant, 835.4, that spontaneously arose within the background. pou5f1 expression is normal in mutant embryos, but sox32 expression is reduced and sox17 expression is lost. Downstream endoderm genes of sox17 are also lost and as a result no endodermal organs develop. Rescue experiments indicate that the mutation is located between sox32 and sox17 in the endoderm pathway. We currently have not been successful at mapping this mutation and therefore are unable to rule out the possibility that it lies in the sox17 gene. However, our data suggest that the mutation occurs in a new gene that is necessary for sox17 expression, potentially working with sox32 and/or pou5f1.

Adipose tissue inflammation has an impact on liver health and it has been demonstrated that chronic alcohol consumption leads to the expression of pro-inflammatory markers in the adipose tissue. A thorough characterization of alcohol-induced adipose inflammation is lacking, and is important to understand in order to identify immune-related mechanisms that drive this phenomenon. Current therapeutic regimens for alcoholic liver disease are ineffective. It is critical to understand how other organs influence liver injury in this disease when developing novel and effective therapies in the future. Alcoholic liver disease exhibits a sexual dimorphism; women are more susceptible to liver injury than men and the same paradigm exists in rodent models. Here, I demonstrate that female mice have greater alcohol-induced adipose tissue inflammation than male mice, evidenced by greater expression of pro-inflammatory cytokines and cell markers. Further, female mice also exhibit higher expression of toll-like receptor genes in the adipose tissue, suggesting a potential role for the innate immune system in alcohol-induced adipose inflammation. Toll-like receptor 4 (TLR4) has been demonstrated to drive inflammation in both the liver and adipose tissue. I used both germline and conditional knockouts of Tlr4 to characterize alcohol-induced changes in the immune cell composition of adipose tissue. Alcohol increased the number of pro-inflammatory adipose tissue macrophages. This macrophage phenotype switching is partially dependent on TLR4; germline, but not myeloid-specific, Tlr4-deletion prevents macrophage phenotype switching. Overall, my work demonstrates that alcohol-induced adipose tissue inflammation is related to liver injury and that TLR4 contributes to adipose macrophage phenotype switching.

Background To investigate plasma microRNA (miRNA) profiles indicative of hepatotoxicity in the setting of lethal acetaminophen (APAP) toxicity in mice. Methods Using plasma from APAP poisoned mice, either lethally (500 mg/kg) or sublethally (150 mg/kg) dosed, we screened commercially available murine microRNA libraries (SABiosciences, Qiagen Sciences, MD) to evaluate for unique miRNA profiles between these two dosing parameters. Results We distinguished numerous, unique plasma miRNAs both up- and down-regulated in lethally compared to sublethally dosed mice. Of note, many of the greatest up- and down-regulated miRNAs, included, but were not limited to, 574-5p, 466g, 466f-3p, 375, 29c, and 148a. There was a statistically significant increase in alanine aminotransferase levels in the lethal compared to sublethal APAP dosing groups at the 12 h time point ( P < 0.001). There was 90% mortality in the lethally compared to sublethally dosed mice at the 48 h time point ( P = 0.011). Conclusion We identified unique plasma miRNAs both up- and down-regulated in lethally dosed APAP poisoned mice.

Chronic intake of alcohol can result in a range of pathology in the liver. Whilst the earliest changes observed with chronic ethanol, including the accumulation of lipid, or steatosis, are readily reversible upon cessation of alcohol consumption, longer exposure to ethanol may achieve more complex disease states including steatohepatitis, fibrosis, and cirrhosis that can cause irreversible damage and progress to fulminant hepatic failure. A key concept in the pathogenesis of alcoholic liver disease is that chronic ethanol primes the liver to increased injury through an interplay between hepatocytes and non-parenchymal cells, chiefly immune cells, of the liver. These relationships between hepatocytes and non-parenchymal cell types in alcoholic liver disease are reviewed in Chapter 1A. The Hypoxia Inducible Factors are a set of transcription factors that classically have been described as affecting a homeostatic response to conditions of low oxygen tension. Alcoholic liver disease is marked by increased hepatic metabolic demands, and some evidence exists for increased hepatic tissue hypoxia and upregulation of hypoxia-inducible factor mRNA with chronic alcohol. However, the biological significance of these findings is unknown. In Chapter 1B, we review the literature on recent investigations on the role of hypoxia inducible factors in a broad array of liver diseases, seeking to find common themes of biological function. In subsequent chapters, we investigate the hypothesis that a member of the hypoxia inducible- factor family, HIF1α, has a role in the pathogenesis of alcoholic liver disease. In Chapter 2, we establish a mouse model of alcoholic liver disease and report data confirming HIF1α activation with chronic ethanol. We demonstrate that HIF1α protein, mRNA, and DNA binding activity is upregulated in ethanol-fed mice versus pair-fed mice, and that some upregulation of HIF2α protein is observable as well. In Chapter 3, we utilize a mouse model of hepatocyte-specific HIF1α activation and demonstrate that such mice have exacerbated liver injury, including greater triglyceride accumulation than control mice. Using cre-lox technology, we introduce a degradation resistant mutant of HIF1α in hepatocytes, and after four weeks of ethanol feeding, we demonstrate that mice with the HIF1α transgene have increased liver-weight to body weight ratio and higher hepatic triglyceride levels. Additionally, several HIF1α target genes are upregulated. In Chapter 4, we examine the relationship between HIF1α activation and hepatic lipid accumulation using a recently published in vitro system, in which lipid accumulation was observed after treating Huh7 cells with the chemokine Monocyte Chemoattractant Protein-1 (MCP-1). We report that MCP-1 treatment induces HIF1α nuclear protein accumulation, that HIF1α overexpression in Huh7 cells induces lipid accumulation, and finally, that HIF1α siRNA prevents MCP-1 induced lipid accumulation. In Chapter 5, we use mouse models to investigate the hypothesis that suppression of HIF1α in hepatocytes or cells of the myeloid lineage may have differing effects on the pathogenesis of alcoholic liver disease. We find that ethanol-fed mice expressing a hepatocyte-specific HIF1α deletion mutant exhibit less elevation in liver-weight body ratio and diminished hepatic triglycerides versus wild-type mice; furthermore, we find that challenging these mice with lipopolysaccharide (LPS) results in less liver enzyme elevation and inflammatory cytokine secretion than in wild-type mice. In Chapter 6, we offer a final summary of our findings and some directions for future work.