The liver is a multifunctional organ that regulates important processes such as lipid, glucose and xenobiotic metabolism. Most of the hepatic functions are implemented in the hepatocytes, which make up the largest cellular proportion of the liver. Various signaling pathways are required for the liver to perform its diverse metabolic tasks, two of them are the Hedgehog (HH) and mechanistic target of rapamycin (mTOR) signaling pathways. Dysregulation of these signaling pathways leads to various liver diseases, such as non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). In order to develop appropriate treatments for these disorders, it is important to understand the molecular interactions of the various signaling pathways involved in their development and progression. However, another aspect is particularly important in the treatment of hepatic diseases - the sexual dimorphism of the liver. A great variety of genes are differentially expressed in the liver of males and females. Signaling pathways affected by hepatic sexual dimorphism include lipid and drug metabolism. Although the influence of sex-specific dimorphism in the liver on diseases and drug doses is well known, there are currently only a few sex-specific treatment concepts. In order to incorporate this aspect in the development of new and more effective treatments, it is important to understand the molecular mechanism and characteristics of sexual dimorphism in model systems. Primary hepatocytes are often used for in vitro analysis of pharmaceutical studies. However, little is known about the development of sexual dimorphism during the culture of primary cells. To facilitate the development of novel sex-specific treatment approaches, the present thesis investigated the interaction of the HH and mTOR signaling pathways in hepatocytes, as well as the development of sexual dimorphism during the culture of primary hepatocytes for up to 96 h. Male and female mice with a hepatocyte-specific knock-out (KO) of the HH signaling were used to examine a possible interaction of HH and mTOR signaling. The repression of the HH signaling cascade leads to reduced mRNA expression of molecules involved in mTOR Signaling as well as decreased mitochondrial adenosine triphosphate (ATP) production in hepatocytes from female KO mice. In contrast, hepatocytes from male KO mice showed reduced phosphorylation of mTOR molecules. Furthermore, an impaired autophagy was detected in primary hepatocytes from both sexes. Consequently, the hepatocyte-specific HH-KO leads to decreased mTOR activity in males and females. However, the KO shows different points of action depending on the sex of the mice. To investigate the crosstalk of the two pathways in more detail, primary hepatocytes from male and female C57BL/6N mice were incubated with the HH inhibitor cyclopamine and the mTOR inhibitors rapamycin and Torin. Thereby, a synergistic effect of cyclopamine and rapamycin could be demonstrated on the repression of the mTOR signaling cascade and mitochondrial ATP production. Although primary hepatocytes have been isolated from male and female mice for all experiments, the further development of the sexual dimorphism of the hepatocytes in monolayer-cultivation is unclear. Therefore, cells from male and female C57BL/6N mice were isolated to conduct transcriptome, proteome and metabolome studies directly after isolation and after 24, 48, 72 and 96 h of cultivation. It was striking that the expression pattern of sexually differentiated genes developed differently during cell culture compared to the translation pattern of sexually differentiated proteins. While mRNA expression showed a large shift after 24 h, there was a feminization of the translation pattern at the protein level throughout the cultivation period. Furthermore, the study revealed a change in the sex-specific gene expression of xenobiotic metabolism with a significant decrease of female-specific expression of Cytochrome P450 (Cyp) 2b13 and Cyp2b9. Since no sex hormones or growth factors were added to the cell culture, a reduction in sex differences during the time of cell culture was to be expected. Instead, we found an increase in sex-specific gene expression for some signaling pathways, such as serotonin and melatonin degradation. For the expression of genes related to androgen signaling, beta-oxidation, hepatic steatosis and amino acid degradation, even a reversal of the sex-specific gene expression could be shown. In conclusion, this investigation reveals a connection between HH and mTOR signaling in hepatocytes. The repression of the HH pathway modulates mTOR signaling and results in impaired oxidative phosphorylation and autophagy. It is conceivable that the synergistic effect of HH and mTOR inhibition could be applied in targeted therapies. Furthermore, the culture of male and female primary hepatocytes demonstrate that sex differences change significantly over time. This should be considered when using primary hepatocytes for initial preclinical studies to analyze potential new drugs. Especially, the alterations in xenobiotic metabolism can have serious effects on pharmacodynamic processes. In addition, the enhancement or reversal of sex-specific expression of some genes in vitro suggests that there must be previously unknown mechanisms that modulate sexual dimorphism independently of the steroids and growth hormones known to date.:Table of Contents 1. Introduction 1.1 Characteristics of the liver 1.2 The hepatic sexual dimorphism 1.3 Interaction of signaling pathways in the liver 1.3.1 The Hedgehog pathway 1.3.2 The mTOR pathway 1.3.3 The role of HH and mTOR in diseases 1.4 Aim of the study 2. Publications 2.1 Cyclopamine and rapamycin synergistically inhibit mTOR signaling in mouse hepatocytes, revealing an interaction of hedgehog and mtor signaling in the liver 2.2 Sex-dependent dynamics of metabolism in primary mouse hepatocytes 33 3. Summary of the thesis 4. References 5. Attachments 5.1 List of abbreviations 5.2 Supplemental Material 5.2.1 Cyclopamine and rapamycin synergistically inhibit mTOR signaling in mouse hepatocytes, revealing an interaction of hedgehog and mtor signaling in the liver 5.2.2 Sex‑dependent dynamics of metabolism in primary mouse hepatocytes 5.3 Description of the author's own contribution 5.3.1 Cyclopamine and rapamycin synergistically inhibit mTOR signaling in mouse hepatocytes, revealing an interaction of hedgehog and mtor signaling in the liver 5.3.2 Sex‑dependent dynamics of metabolism in primary mouse hepatocytes 5.4 Statement of Authorship 5.5 Curriculum vitae 5.6 Publications 5.7 Acknowledgement

The sexing of human skeletal remains is important for identification and demographic purposes. It is made more difficult when elements such as the skull and pelvis are not recovered or are in too poor of a condition to assess. Previous studies have used carpal (wrist) bones of contemporary populations to assess the viability of these skeletal elements exhibiting sexual dimorphism, as these bones are small, compact elements that are usually recovered in good condition. This study evaluates the use of carpal bones recovered from an ancient Maya population from Belize to determine the biological sex of individuals. The study sample is part of the Maya Archaeological Skeletal Collection (MASC), which contains individuals from the sites of Lamanai, San Pedro, Altun Ha, and Marco Gonzalez and dates from the Late Maya Pre-Classic (400 BC-AD 250) to the Spanish Colonial period (AD 1521-1821). Multiple measurements were taken on 36 capitate, 34 lunate, 34 scaphoid, 27 trapezium, 24 hamate, 22 triquetral, 22 trapezoid, and 16 pisiform bones from several individuals. Discriminant function analysis was used to determine if sexual dimorphism is measurable in this population using these elements. Previous studies used populations with known identities, assessing individuals from crypts, graveyards, or medical collections from the last few centuries. This study varies from previous studies as it utilizes archaeological remains, making this study one of the first to evaluate non-contemporary remains with unknown sex. Results of this study demonstrate that this population exhibits sexual dimorphism and discriminant function analysis can be used to distinguish between two groups. This demonstrates that carpals could be used to help determine biological sex of archaeological populations as well as a tool to help with identification in forensic cases.

The distribution of dioecious species among forty-four plant communities of western United States was examined. The dioecious habit is most prevalent in harsh environments. In many communities, over 20 percent of the species and 40 percent of the individuals are dioecious. Dioecy is most common among woody species which are pollinated by wind. It is concluded that inbreeding depression alone is insufficient to account for all known facts concerning dioecy. Disruptive selection acting upon differential success of gametes produced on sites of differing quality appears to have played a major role in producing separate sexed individuals. Dioecious species reported to exhibit sex reversal and factors believed to promote such reversals are tabulated. It is concluded that for dioecious species which exhibit differential resource utilization by the sexes, individuals which produce offspring capable of sex reversal have a selective advantage. Sex reversal provides a strategy that permits dioecious species to reproduce optimally in patchy environments.

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Sexual dimorphism, i.e., differences in morphology, physiology, and behavior between conspecific males and females, is ubiquitous, extensive, and often species-specific, indicative of its rapidly evolving nature. Ever since Darwin first described a general theory of sexual selection to explain the extraordinary differences between males and females of the same species, biologists have proposed a variety of mechanisms ranging from runaway selection to good genes to sexual conflict. While a popular approach is studying the effects of sexual selection on different components of fitness, the results of these studies are generally difficult to interpret and are typically not generalizable across populations, let alone taxa. Recent advances in the “omics” field are transforming the way that we study patterns and processes involved in sexual selection. At the molecular level, sexual dimorphism is present in gene expression differences between the sexes, providing a powerful framework to study sexual selection. By studying genes that are sex-biased in expression, we will better understand the underlying genetic basis of traits that are sexually dimorphic. Alreadly, studies of sex-biased genes in model organisms, particularly Drosophila, have revealed that male-biased genes are among the most rapidly evolving functional classes of genes. However, while a number of intrinsic factors appear to correlate with evolutionary rate (e.g., gene expression level, codon bias), it is unclear whether any of these factors drive the rapid divergence of male-biased genes. Another important discovery is the prevalence of sex-biased gene expression. However, even with widespread sexual dimorphism at the phenotypic level, it remains unknown the extent to which sex-biased gene expression exists in humans and their primate relatives. In fact, studies of sexual dimorphism on a molecular level in primates have been very few, even though understanding this phenomenon in humans could further our knowledge of the nature of sex-biased phenotypes and diseases. In this thesis, I advance our knowledge of the genetic bases and mechanisms that shape sexual dimorphism. First, I review a classic framework that biologists have traditionally applied to define and partition fitness measures between males and females in the model system, Drosophila. Second, I apply a molecular framework to compare the relative roles of intrinsic factors on the evolutionary rate of rapidly evolving male-biased genes in Drosophila. Third, I review the current state of our knowledge of sexual dimorphism and sex-biased gene expression in humans. Fourth, I present a bioinformatics framework to identify the extent of sex-biased expression in primate tissue and to examine the selective forces involved in their evolution. Overall, I demonstrate the effectiveness of using a functional comparative genomics approach in studying the nature of sexual dimorphism at the molecular level across multiple taxa. / Biology

Pink heelsplitter (Potamilus alatus) mussels collected from Kentucky Lake, TN were held at two bottom locations (0.6 m, 2.5 m) and suspended in pocket nets (at depth about 1.0 - 1.5 m) in a pond at the Freshwater Mollusk Conservation Center (FMCC), Virginia Tech, for 1 yr. Survival of mussels after 1 yr was significantly different, with poorest survival (30 %) in the bottom of the deep end; and no difference between the shallow end (83.3 %) and the suspended pocket nets (63.3 %). Survival of mussels was inversely related to water temperature (r = - 0.72); lowest monthly survival occurred in summer, resulting in a significant difference among the three locations with a similar trend after 1 yr. The glycogen reserves of mussels in captivity for 1 yr differed by pond location, higher in mussels at the shallow end than those in suspended pocket nets and at the deep end. Therefore, the shallow end of pond was more suitable for holding mussels long-term, while the suspended pocket nets are an alternative site for holding captive mussels. Additionally, dissolved oxygen was very low at the deep end (1.9 mg/L) in summer, while it was adequate (range from 5.7 - 6.4 mg/L) at the location of suspended pocket nets, and 5.0 mg/L at the shallow end (24.7 °C). Data for 40 specimens indicated that sexual dimorphism in valve shape occurred in P. alatus. Female mussels had a significantly (p < 0.0001) greater ratio of height (H) to length (L) (52.3 %) and width (W) to length (31.8 %) than males (H/L: 48.4 %; W/L: 28.8 %), respectively. The posterior ends were somewhat round to oval in males and bluntly squared or truncated in females. Female mussels were more inflated than males. These morphological differences can be used to distinguish females from males during field collections. The red drum (Sciaenops ocellatus) was identified as a new fish host for P. alatus, as 48 active juveniles were transformed by this species, which is not a natural host. Four glochidia were observed on the fins versus 2,307 on the gills of five red drum. Freshwater drum also was verified as a suitable host fish, but black crappie (Pomoxis nigromaculatus), banded sculpin (Cottus carolinae), yellow perch (Perca flavescens) and nile tilapia (Oreochromis nilotica) did not support transformation of glochidia to juveniles. Survival and growth of propagated juveniles of P. alatus were assessed regarding the effects of algal diets (Nannochloropsis oculata and Neochloris oleoabundans) and substrate type (fine sediment and sand). Overall, survival of juveniles after 17 d ranged from 23.8 to 66.8 %, with mean of 48.5 %; however, survival dramatically declined during the next 2 wk period to only 5.8 % (range of 1.8 to 7.8 %). Survival rate of juveniles was significantly different (p = 0.027) between substrates, but not in diets (p = 0.520), with the lowest survival rate of 23.8 % in sand substrate and fed N. oculata. Juveniles grew faster in fine sediment (23.0 % increase in shell length) than in sand substrates (10.5 % increase) (p = 0.002). Moreover, mean growth rate of juveniles was 4.9 &#956;m/d during the first 2 wk, but decreased to 0.2 &#956;m/d in the remaining 2 wk. Therefore, fine sediments seemed more appropriate for juvenile culture compared to sands. Both species of algae, N. oculata and N. oleoabundans, can be used to feed juveniles in the laboratory. Adult pink heelsplitters were used to study feasibility of pearl production by using two surgical implants (non-nucleated implant = NNI, and image pearl implant = IPI) in two ponds of different nutrient levels (FMCC pond and Duck pond). NNI and IPI pearls with purple or purplish luster were successfully produced in P. alatus. Pearl weight was not significantly different (p = 0.562) between two ponds. No differences in monthly survival rates of mussels were observed in either pond (p = 0.051), or among mussels with surgical implants and the no-surgery control mussels (p = 0.881). Consequently, P. alatus can be considered a potential species for producing purple pearls in pearl culture. Additionally, mussels in the Duck pond had higher (p < 0.0001) glycogen levels, similar to those in wild collected mussels, than those in the FMCC pond, indicating that this pond environment may be more suited for holding implanted mussels in captivity. / Master of Science

The placenta plays a vital role in a healthy pregnancy by supporting the intricacies of fetal development. Over 10% of pregnancies experience impaired placental function, resulting in the loss of critical neuroactive steroids the fetal brain cannot yet make, thus leaving them vulnerable to perinatal brain injury and abnormal neurodevelopment. However, this vulnerability is not always equal. Many neurodevelopmental disorders exhibit a sex bias in incidence and severity. I hypothesize that loss of placental support during pregnancy results in sex differences in both behavioral presentation as well as on the cellular and transcriptomic levels. Utilizing the akr1c14cyp19aKO (plKO) mouse model, which features placenta-specific allopregnanolone (ALLO) knockdown, I investigated the sex specific impact of placental hormones on cerebellar development. Here I show that placental ALLO is essential for cerebellar white matter development and inflammatory regulation via microglial function. Male mice without placental ALLO exhibit signs of placental inflammation, accelerated postnatal myelination, and defects in microglial phagocytosis of excess myelin. Alternatively, females seem to be more resilient with a progressive anti-inflammatory profile across development and reduced myelination. Additionally male plKO show autism-like behaviors such as deficits in social behavior and increased stereotyped behavior. The females do not exhibit this phenotype. My main goals were threefold; to investigate how male and female inflammatory profiles differ and where this difference originates, to investigate how this inflammation impacts microglia and thereby oligodendrocytes, and how I can alter microglial function in a way to improve plKO outcomes. Mechanistically, these changes appear to be in part due to baseline sex differences in response to inflammatory stimuli which prime microglia to differentially support the surrounding white matter. Together, this work supports a novel link between placental ALLO loss, microglial function, and sex specific presentation of neurodevelopmental disorders.

The Tardigrada are hydrophilous, segmented, molting micrometazoans that occupy a diversity of niches in freshwater, marine, and terrestrial habitats. A sister group of the arthropods, this phylum of bilaterally symmetrical lobopods, most less than 1 mm in length, have a hemocoel, a complete digestive tract, a dorsal gonad with one or two gonoducts, and a dorsal lobed brain with a ventral nerve cord and five ganglia. About 1000 species have been described based on the morphology of sclerified structures, especially the claws and buccal-pharyngeal apparatus. Reproduction occurs through fertilized or unfertilized eggs, with individuals being either gonochoric, unisexual, or hermaphroditic, and eggs are deposited either freely or within the shed exuvium. Parthenogenesis, very frequent in limnic and terrestrial tardigrades, allows them to colonize new territories by passive dispersal of a single individual. Quiescence (cryptobiosis: anhydrobiosis, anoxybiosis, cryobiosis, and osmobiosis) and diapause (encystment and resting eggs) occur during the tardigrade life history. Ecological parameters and global distribution patterns are poorly known or understood. Methods for collection, microscopy, and culturing have been developed.