Using in-depth interviews with 20 transgender and gender non-conforming (TGNC) young adults (aged 18-24) in Florida, I examine how TGNC people navigate accountability structures to meet gendered expectations in the US healthcare system. While a growing body of sociological literature has expanded West and Zimmerman's "doing gender" and social accountability concepts, gaps remain in understanding how transgender populations encounter accountability structures, and the navigation of such structures by TGNC youth. Utilizing thematic analysis, I examine how TGNC people manage a practice I call "cisnormative accountability." Specifically, I demonstrate how the majority of respondents were often held accountable to a cisgender standard of accessing and utilizing health services through the gendered expectations of 1) enforcing a cisnormative standard of care through medical notions of binary gender and 2) upholding the medical model of transgender identity. Further, I outline the process of and consequences of "cisnormative accountability" and its potential implications for sociological studies of transgender experiences in healthcare.

Polarized cell growth is a fundamental biological process that is tightly regulated spatially and temporally. In plants the key systems to study polar cell growth are pollen tubes and root hairs. In recent years a lot of work has focused on elucidating the mechanisms that mediate this process. The actin cytoskeleton plays a key role in polarized cell growth. Different studies in plant and animal models show that signaling mediated through small GTP-binding proteins is a common theme in actin signaling. In recent years many groups have shown that small GTP binding proteins regulate actin dynamics through the activity of Actin Binding Proteins (ABP). In this study I explored the function of two ABPs from Arabidopsis Thaliana: Actin depolymerizing factor (ADF) and a novel actin-binding guanine exchange factor (GEF). I used Arabidopsis protoplasts as a system to study the function of these proteins. We showed through over-expression of the GFP labeled GEF8 under the constitutively active 35 S promoter, that GEF8 labels the prominent cable like structures inside the cell. Using actin and tubulin binding drugs such as Latrunculin and Oryzalin we showed that GEF8 labels actin cables. Using the Yeast 2 Hybrid system we determined that GEF8 binds actin filaments directly. We established that GEF8 interacts with actin through the unique N terminus of the protein. Finally, using the Basic Local Alignment Search tool we showed that the N terminus of GEF8 is homologous to the Actin Binding Protein 140, a well-established protein marker in Yeast. ADF is an established key regulator of the actin cytoskeleton. Much is known about ADF regulation in animal systems. In plants it has been shown that the small Rho type GTP binding proteins, called RAC/ROPS, regulate ADF activity and that overexpression of RAC/ROPs causes the inactivation of ADF through the phosphoryaltion on Serine 6. However, little is known about the proteins that transduce the signal from small GTP binding proteins to the ADF. Here we show some evidence that upon overexpression of Ric 4 (a RAC/ROP effector known to play a role in actin polymerization), ADF gets displaced from the filament. Moreover, ADF is known to be inactivated by phosphorylation at Ser6; the kinase responsible for this phosphorylation has not been identified in plant. We observed that over-expression of Calcium Dependent Protein Kinase 16 (CDPK16) in protoplasts also induced dissociation of ADF from actin cables. These results suggest that both of RIC4 and CDPK16 may play a role in the pathways that regulate ADF activity.

Protein phosphatases regulate a wide array of proteins through post-translational modification and are required for a plethora of intracellular events in eukaryotes. While some core components of the protein phosphatase complexes are well characterized, many subunits of these large complexes remain unstudied. Here we characterize a murine loss-of-function allele of the protein phosphatase 1 regulatory subunit 35 (Ppp1r35) gene. Homozygous embryos lacking Ppp1r35 initiate developmental delay beginning at E7.5 and have obvious morphological defects at slightly later stages. Mutant embryos do not initiate turning and fail to progress beyond the size and relative development of an E8.5 embryo. Consistent with recent in vitro studies linking PPP1R35 with the microcephaly protein Rotatin and a role in centrosome elongation, we find that the Ppp1r35 mutant embryos lack primary cilia. Histological and molecular analysis of Ppp1r35 mutants revealed that notochord development is irregular and discontinuous and that the floor plate of the neural tube is not specified, consistent with defects in primary cilia. Similar to other mutant embryos with defects in centriole function, Ppp1r35 mutants display increased cell death that is prevalent in the neural tube and an increased number of proliferative cells in prometaphase. We hypothesize that loss of Ppp1r35 function abrogates centriole homeostasis, resulting in both a failure to produce functional primary cilia and cell death and/or cell cycle delay that leads to embryonic lethality. Taken together, these results highlight the essential function of Ppp1r35 during early mammalian development and implicate this gene as a candidate for human microcephaly.

The objective of this study is to investigate heart rate variability (HRV) over a period of 24 hours with special focus on patterns during the sleep period. Comparisons were made between sleep and the full 24-hour data. The Polar M430 watch was used to record heart rate (HR) data for about 24 hours, and the subject kept a journal of the activities they performed throughout the day. The data was visualized as heart rate in beats per minute (BPM) as a function of time. To check the accuracy of the Polar watch, a 7-minute recording of HR from the watch was performed simultaneously with a gold standard (IX-TA 220 and IX-ECG12, iWorx, Dover, NH). The data was downloaded into Excel spreadsheet, then analyzed. Analysis included calculations such as: standard deviation, range, mean, and the Fast Fourier Transform (FFT). HR data analysis was performed for the 24hr and sleep period. Since daily activities (which can affect HR) were difficult to control in the study subjects, the sleep period was chosen for more detailed analysis. A trend of lower HR and HRV was seen during sleep. Although the watch had modest accuracy, it was able to show some HR patterns during sleep. FFT analysis of the sleep data suggested existence slow HR cycles(which corresponds one cycle every > 5 minutes).

The central dogma of biology states that genetic information describes the flow ofinformation from DNA to RNA and then finally resting in proteins. The fundamental aspect that underlies all aspects of life is the expression and modification of proteins. One can even argue that there is no life without proteins. As such, many human diseases are either directly or indirectly related to dysfunction of proteins and can potentially be solved through protein therapeutics. Consequently, scientists have begun to harness the diversity of proteins to treat the diseases which plague man in the form of protein therapeutics such as clotting factors, cytokines, and growth factors. Unfortunately, the short circulation half-life of proteins is a major limiting factor which must be overcome before their widespread adoption as a platform for therapeutic development. Contributing factors to this short circulation half-life include renal elimination, proteasomal degradation, and metabolism in the liver. Namely, renal elimination is the main challenge for protein therapeutics and warrants clinicians to resort to higher doses and more frequent administrations to maintain necessary concentrations in the body. Unfortunately, side effects of this approach are dose limiting toxicities and reduced therapeutic outcomes as drug concentrations fluctuate drastically. As a result, addressing the challenge of renal elimination for protein therapeutics would allow for the development of novel treatments which were previously not viable. The human glomerulus readily filters out any particle smaller than approximately 30 kDa in weight. As a result, strategies adopted all share a common theme of endowing the protein with a greater effective size without compromising their natural activity against the intended target. The current approaches include conjugation to a polymer (i.e., PEGylation), covalent or non-covalent binding to a larger protein, or conjugating to the neonatal Fc receptor. Major limitations of these approaches include compromised activity caused by steric hinderance rooted inconjugation to moieties of larger size. This issue applies to all of the aforementioned reported approaches wherein activity becomes reduced, thus necessitating higher dosages. Furthermore, other limitations also exist such as humoral immune responses against polymers through anti-PEG antibodies, occurrence of organ damage, and solubility issues. A novel approach was recently developed by the Alhamadsheh lab which demonstratedthe ability of a small molecule linker termed “Transthyretin Ligand for Half-life Extension” (TLHE) to extend the circulation half-life of Gonadotropin releasing hormone. Most essentially, this was accomplished without compromising the potency or introducing a major sterically bulky group to the original peptide. Furthermore, additional concerns such as solubility issues was demonstrated to not be an issue either. In this work, human Interleukin-2 (IL-2) was chosen as a proof of concept to demonstrate application of the TLHE technology in a protein to address the aforementioned half-life challenge. Previously, a mixture of IL-2 and TLHE-IL-2 was demonstrated to maintain comparable activity to control IL-2 in both in vitro and ex vivo efficacy assays. Furthermore, a pharmacokinetic evaluation in rodents demonstrated significant half-life extension of TLHE-IL-2. The objective of this work was to shift the ratio of the IL-2/TLHE-IL-2 mixture to majority TLHE-IL-2 and or enhance the yield of the mixture for further in vivo efficacy evaluation. What would happen to a viral infection if suddenly there were a billion fake receptors forevery real target receptor. A version of this question is what led to the development of a novel HIV therapy at Duke University around 1996. Fast forward more than 30 years, man still lacks the proper tools to combat viral infections. One can argue that the Achilles’ heel of viral infections is their need to bind a specific receptor. This protein-protein interaction between viral proteins and human receptors is arguably the fundamental point behind all viral infections. Recently, the COVID-19 pandemic again challenged man to develop new weapons at a revolutionary pace in order to save lives. During this time, the Pentelute lab at Massachusetts Institute of Technology reported a humanized version of the peptide sequence thought to represent the binding face of the human ACE2 receptor41. The 23 amino acid sequence was derived from the α1 helix of ACE2 peptidase domain and referred to as, spike-binding peptide 1 (SBP1). It was this sequence which was postulated to be responsible for binding the receptor binding domain of the notorious COVID-19 spike protein. However, a major limitation of peptide is their short in vivo half-life (through serum proteases and renal filtration). Therefore, the main aim of our proposed research was to employ the TLHE approach to extend the in vivo half-life of the SBP1 peptide. This would allow the creation a COVID-19 entry inhibitor that could help combat the COVID-19 pandemic.

Gamma-hydroxybutyric acid (GHB) is an endogenous shorty chain fatty acid that is used clinically as Xyrem to treat narcolepsy. GHB is best known for its illicit use and abuse due to its sedative/hypnotic and euphoric effects. It is used in body building, for recreational use and sexual assault. Nonlinear toxicokinetics of GHB has been described in humans and rats with decreased total clearance at higher doses due to capacity-limited metabolism resulting in a higher plasma exposure. Renal clearance increases with increasing dose and becomes the major route of GHB elimination in overdose cases due to saturation of GHB metabolism. Proton- and sodium-dependent monocarboxylate transporters (MCTs (SLC16A) and SMCTs (SLC5A)) have been identified as major transporters in the renal reabsorption of GHB moving it from filtrate back to systemic circulation. Our laboratory has previously investigated sex differences in GHB toxicokinetics at 600 mg/kg in rats and identified sex differences in MCT expression in the liver and kidney. These data suggest that individual sex hormones may be involved in altering MCT and SMCT expression in drug disposition tissues and as a result alter GHB toxicokinetics. The present study had three objectives:1) GHB toxicokinetics were evaluated over the estrous cycle, and in the presence and absence of sex hormones following a dose of 1000 mg/kg iv in rats; 2) The role of individual female sex hormones on altering GHB toxicokinetics was investigated at 1000 mg/kg and 1500 mg/kg iv following sex and cross-sex hormone treatment with 17β-estradiol and progesterone, alone or in combination; and 3) renal MCT1, MCT4, SMCT1 and CD147 mRNA and membrane protein expression were quantified in response to female sex and cross-sex hormone treatment. We have demonstrated that GHB toxicokinetics and renal clearance vary between sexes, over the estrus cycle in females and in the absence of female and male sex hormones. In hormone-treated animals, GHB toxicokinetics were altered following sex and cross-sex hormone treatment with significantly increased total clearance and decreased GHB plasma exposure at 1000 mg/kg. Significant differences in renal and metabolic clearance were observed following 1000 mg/kg and 1500 mg/kg GHB suggesting altered regulation of the underlying clearance pathways. Additionally, we have investigated the renal mRNA and membrane-bound protein expression of MCT1, MCT4, CD147 and SMCT1 which were significantly altered in response to female sex hormones in both OVX (ovariectomized female rats, ovary removal surgery performed) and CST (castrated male rats, testicles removal surgery performed) rats. The mechanisms underlying MCT/SMT regulation by female sex hormones appear to vary based on the specific transporter. The alteration of renal monocarboxylate transporters in response to female sex hormones may contribute to the observed differences in GHB toxicokinetics, which may benefit the potential antidotes of GHB as a combined therapeutic strategy in clinic. In future, inhibition studies should be performed with the coadministration of MCTs/SMCTs inhibitor with GHB to further confirm the contribution of monocarboxylate transporters to GHB toxicokinetics following sex and cross-sex hormone treatment. The influence of female sex hormones on GHB-related metabolic enzymes and monocarboxylate transporters in liver should be evaluated to further explore the mechanism of underlying the observed alterations in metabolic clearance. Sex hormone receptor expression should be evaluated by western bolt and correlated with transporter expression, combined with analysis of sex and cross-sex hormone replacement with coadministration of sex hormone receptor antagonists, to further elucidate the mechanisms underlying MCTs/SMCTs regulation in response to female sex hormones. Additionally, GHB TK studies should also be conducted, combined with sex and cross-sex hormone replacement with coadministration of sex hormone receptor antagonists, to further confirm the effect of sex hormone receptor antagonist in GHB toxicokinetics. The MCT/SMCT expression is a key determinant of their substrates’ drug disposition; sex differences and altered regulation in response to sex and cross-sex hormone treatment may contribute to differences in GHB toxicokinetics and toxicity.

The aryl hydrocarbon receptor (AHR), a cytosolic ligand-activated transcription factor, has been acknowledged as a critical regulator of xenobiotic-induced toxicity and carcinogenesis. In the absence of ligand, the AHR is cytoplasmic in a complex with Hsp90, p23, XAP2, and Src. The AHR complex translocates to the nucleus upon ligand binding. After releasing its chaperones, it forms a heterodimer with ARNT, which subsequently binds to a dioxin-responsive element (DRE) for target genes transcription. Multiple aspects of cells are altered by the substantial expression of AHR target genes. Even without the AHR ligand, the cytoplasmic AHR plays a critical role in tumor progression by affecting various cellular functions. Thus, understanding the mechanisms of AHR degradation is crucial, which provides novel ways to control the AHR target genes transcription and cellular functions. In addition to the 26S proteasomal degradation triggered by ligand or geldanamycin treatment, we discovered a novel AHR degradation pathway mediated by autophagy-lysosome in A549 cells. Specifically, the chaperone-mediated autophagy (CMA) facilitates the degradation of basal AHR in the lysosome. It can be activated by 6-AN, resulting in downregulated AHR protein levels and functions, including the ligand-dependent target genes transcription and cell migration/invasion process in A549 cells.p23 as a part of the AHR cytoplasmic complex has been continuously studied in our lab over the past decade. The most prominent role of p23 is protecting AHR from degradation in both immortalized cancer cell lines (mouse hepatoma Hepa1c1c7, human hepatoma Hep3B, human cervical HeLa) and untransformed human lung bronchial/tracheal epithelial (HBTE) cell lines. It encouraged us to investigate the mechanisms further. In A549 cells, downregulation of p23 content reduced AHR protein levels, partially due to an elevated AHR protein degradation. This degradation was not reversed by proteasome inhibitor MG132 but partially restored by lysosome inhibitor CQ. We cannot rule out the possibility that selective macroautophagy was involved in the basal AHR degradation in A549 cells since the PLA results showed a positive interaction between AHR and LC3B. So far, Hela cells could be the best expression system for HaloTag-AHR overexpression. Thus, we can use the HaloTag technology as a powerful tool to study the AHR degradation mechanism via protein labeling and LC-MS/MS analysis.

The provision of adequate nutritional care to hospital patients continues to be an international problem, despite numerous initiatives and attempts by interested parties over several decades to make improvements. The focus of this research was to critically evaluate the effects a hospital ward eating environment can have on patients’ foodservice experience and to establish if providing an enhanced dining environment could improve outcomes. A case study approach was employed on two Orthopaedic wards in an Acute Care Hospital which considered the variables that concurred in the contextual environment of the foodservice provision, to provide an in depth appreciation of Orthopaedic patients’ dining experience. The study used a mixed methods, sequential, exploratory, research process, consisting of four phases incorporating; semi structured interviews with patients, patient questionnaires, exploratory interviews with stakeholders and measurement of patients’ food intake and mood. Following the empirical processes this study has engaged in, theoretical contributions have been made which include; (1) A conceptual model for factors affecting patients’ foodservice experience developed from a synthesis of literature and theories with regard to the provision of hospitality, mealtime experiences and nutritional care provision for patients; (2) A questionnaire to measure hospital patients’ overall food experiences has been developed; (3) An explanatory model for factors influencing hospital patients’ foodservice experience has been developed; (4) A comparison of the patients’ mean, daily, nutritional intakes with the recommended levels, provided updated evidence of poor nutrition in the research setting, whilst factors influencing reduced food intakes were established; (5) The provision of an enhanced group dining experience for the patients, established positive outcomes for patients and stakeholders; (6) A theoretical model was developed that establishes a hierarchy of factors influencing Orthopaedic patients’ foodservice experience and food intake. This research study makes a contribution to our understanding of how sociological and environmental factors can enhance patients’ dining experience which may ultimately lead to improved nutritional intake.

Therapeutic footwear is provided to patients with the primary aims of reducing foot pain and improving foot health. Therefore, it is of concern that patients choose not to wear it. This choice has been attributed to its poor appearance. However, there may be other reasons for this choice and the reasons may be more complex than previously acknowledged. Therefore, the aim of this study was to investigate the users’ experiences of this footwear. In the context of this study the ‘users’ are women with rheumatoid arthritis. This study was based on an interpretive phenomenological approach (IPA) that employed conversational style interviews to gather data. Ten women with RA and experience of wearing the footwear were recruited and following ethical approval the interviews were carried out, transcribed, organised and analysed. Five organising themes emerged as findings. These were the participants: • feelings about themselves, • feelings about the footwear, • unmet needs and expectations, • behaviour with the footwear and • feelings about the consultation with the practitioner. The final, global theme was that the patient/practitioner consultation has a powerful influence on the women’s feelings about themselves, the footwear, their expectations of it and their behaviour with it. This study has provided insight into their experiences of this footwear in that, unlike any other intervention, it replaces something that is normally worn and is part of an individual’s perception of self and their perception of how others see them. The footwear impacts more on women’s emotions than previously acknowledged and in addition, it has been found that the consultation influences their emotions and behaviour with the footwear. As well as improvements in footwear design and the service that delivers it, the training of the practitioners in a more patient focussed consultation style could improve the women’s experience and engagement in the footwear as a health intervention, as well as something that is ‘worn’.

Spinal cord injury results in loss of motor function and sensory perception. A myriad of obstacles prevent axonal regeneration and ultimately functional recovery in those afflicted with spinal cord injury. Combinatorial strategies addressing many of these obstacles simultaneously have shown promising results. Laboratories investigating contusional spinal cord injuries must overcome the formation of a fluid filled cyst, a physical gap that axons must traverse, at the injury epicenter. To fill the cyst, our lab has generated a 3-D electrospun matrix that is capable of directing neurite outgrowth, delivering neurotrophic support, and reducing the activity of neuroinhibitory compounds. These electrospun matrices were surgically implanted into female Long Evans Hooded rats aged approximately 60 days using a complete transection model of SCI. Following injury, rats with implants showed greater functional recovery than controls. In Chapter 1, we introduce spinal cord injury, the epidemiology, pathology and potential for regeneration, followed by our novel electrospun implant. Chapter 2 details the materials and methods. In Chapter 3, we relate the functional recovery seen to a histological analysis. The histological analysis consists of three parts: the implant integration into the host, the axons above, in and below the implant, and the functional vascular supply found within the implant. In Chapter 4, we designed a modified implant and discuss the use of this implant in vivo. With our modified implant we were able to demonstrate cellular influx and the generation of a vascular network within the implant, but poor axonal regeneration. Finally in chapter 5, I discuss potential future modifications to our electrospun matrix as well as suggestions to consider for improved functional outcome.