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Hepatobiliary Implications of Acute Loss of Adherens Junctions from Cholangiocytes
No full textCholangiocytes, a heterogeneous population of polarized epithelial cells lining the bile ducts, play critical roles in liver function. Many hepatic pathologies stem from or are associated with disruptions of epithelial polarity and barrier integrity. Despite their importance, cholangiocyte barriers remain poorly understood. While tight junctions, the primary occluding structures, are increasingly acknowledged in liver pathobiology, adherens junctions (AJs) remain largely unexplored, especially in cholangiocytes, despite their crucial roles in adhesion, polarity, and partial contribution to barrier function. β-catenin, a well-characterized transcriptional co-activator in the liver, also functions within AJs, where its loss can be compensated by γ-catenin, a homologous desmosomal protein.
To investigate the consequences of AJ loss, we generated Opn-iCreERT2; Ctnnb1-fl/fl; Jup-fl/fl mice (DKO) to inducibly and specifically delete β- and γ-catenins in cholangiocytes. Following recombination, DKO mice exhibited 100% mortality within 5 weeks, along with severe morbidity characterized by jaundice, weight loss, lethargy, and severe liver injury. Histological analysis revealed biliary infarcts, inflammation, portal fibrosis, and stellate cell activation. CK19-positive bile ducts in DKO livers were irregular, with collapsed or absent lumens, as observed via immunohistochemistry and TEM. Severe intrahepatic cholestasis in DKO mice was evidenced by a tenfold reduction in bile flow rate and elevated bile acid levels in serum and liver. Ink injection studies demonstrated blebbing and bile leakages along the biliary tree. RT-qPCR revealed adaptive changes in bile synthesis, transport, and composition, but these were insufficient to counteract the injury. Single-nucleus RNA sequencing (snRNA-seq) identified altered hepatocyte clustering and a distinct cholangiocyte population, with gene signatures indicating junctional remodeling and upregulated necroptosis. To explore potential repair mechanisms, we induced partial AJ loss with only limited tamoxifen dosing. These mice showed improved survival (55% probability at 6 weeks), with severe hepatobiliary injury at two weeks, but significant recovery by four, and near-complete injury resolution by six weeks post recombination. The presence of A6-positive hepatocytes at a 2-week timepoint suggested hepatobiliary transdifferentiation, potentially contributing to repair.
This work highlights the essential role of AJs in cholangiocyte function and bile duct integrity. Furthermore, our model also provides a unique tool to study various aspects of cholestatic disease pathogenesis
Inflammation and Redox Biology in Pneumonia
No full textOver the course of a lifetime, the average person will experience hundreds of respiratory infections, with the greatest susceptibility occurring at early and advanced ages. While for most individuals, symptoms will remain mild, these infections can induce significant morbidity and mortality across the global population. Acute respiratory infections account for thousands of hospitalizations and billions in associated healthcare costs in the United States alone. Understanding the mechanisms behind why some individuals will require additional care and others remain relatively asymptomatic could lead to a reduction of overall mortality and reduced economic burden for individuals and the healthcare system.
A common complication of respiratory viral infection is secondary bacterial pneumonia, which exacerbates symptoms and increases mortality risk. Current best practice is empiric treatment with antibiotics upon admission to the hospital. With the continued rise of multidrug-resistant bacteria, there is a dire need to generate new therapeutic options and understand mechanisms behind the increased susceptibility to secondary bacterial pneumonia to reduce the antibiotic burden and improve patient outcomes. Additionally, there are currently few antiviral drugs approved for treatment of respiratory viral infection, and those that are, have narrow windows of effectiveness.
The focus of this dissertation is on new methods of predicting respiratory infection etiology and severity within the highly susceptible pediatric population. Using blood biomarkers of inflammation, we can identify those individuals likely to require prolonged care. We could also use these biomarkers to distinguish between confirmed viral or community acquired pneumonia (CAP) cases. In a mouse model of severe respiratory viral-bacterial infection we assessed potential mechanisms of viral-induced secondary bacterial pneumonia through modulation of redox biology and surveyed pharmacological modulators to determine their potential as therapeutic options.
These studies highlight that measurable blood inflammatory responses could be used to determine those patients who will require additional care in response to infection, and that modulation of pathways within the inflammatory response could be useful therapeutics
On the Ground with Community Partners, Researchers and Residents: Learning from One Another to Foster Community Resilience.
No full textThis workshop will focus on the experiences of community partners, residents and interdisciplinary researchers working together on a community-centered violence prevention project (ReCAST-Resiliency in Communities after Stress and Trauma). The intervention uses CBPR methods to engage youth and adults to build collective efficacy and community resilience to prevent youth and community violence and improve community wellbeing. It includes a training program for youth and adults, and a community-based project to address issues identified by young people in the training. Participants learn relationship building strategies, restorative approaches to intervene to prevent violence, and ways to advocate for community resources to support community resilience and safety. In this session, researchers along with partners, facilitators and residents from our Sto-Rox community will discuss what we have learned from one another about ways to foster authentic community engagement, build on existing efforts, integrate residents’ lived experiences, focus on community strengths, and elevate youth voice
Data associated with publication: "The Difficulty of Measuring Surface Topography in Additive Manufacturing: A Comparison Between Measured and True Surface Features of Binder-Jet Printed Samples." by C. Brown, M. Nescio, V. Chadha, C. Zheng, E. Abelev, M. Chmielus, T. D. B. Jacobs, published in the ASME Journal of Tribology, 2025
No full textSurface topography represents a critical barrier to the advancement of additive manufacturing (AM). Because some internal features cannot be polished, and because of a growing trend of in situ process monitoring, it is important to understand the as-built surface topography of AM components. Here we highlight the challenges of using industry-standard surface-measurement techniques on binder-jet-printed parts. We measured the topography of binder-jet-printed Inconel Alloy 625 samples in their green state and over the course of sintering; this system allowed the investigation of identical starting materials undergoing systematic changes in topography. Specifically, we compared the results from industry-standard surface-measurement techniques— optical interferometry, 3D microscopy (by fringe projection), and stylus profilometry—against the “true topography,” as revealed by cross-section scanning electron microscopy. While the true topography changed significantly with sintering, the industry-standard techniques detected no change in root-mean-square height because of complex surface features, including multi-scale topography, overhangs, and steep surface slopes. While these findings do not invalidate the use of industry-standard techniques for binder-jet-printed samples, they demonstrate a challenge in their application, and they motivate the development of new metrics and new techniques to more accurately describe surface topography in AM
Understanding the role of UV-DDB in the SMUG1-mediated repair of oxidative DNA damage
No full textUV-damaged DNA-binding protein (UV-DDB) is a heterodimeric protein, consisting of DDB1 and DDB2 subunits, that works to recognize DNA lesions induced by UV damage during global genome nucleotide excision repair (GG-NER). Our laboratory previously discovered a non-canonical role for UV-DDB in the processing of 8-oxoG, by stimulating 8-oxoG glycosylase, OGG1, activity 3-fold, MUTYH activity 4-5-fold, and APE1 (apurinic/apyrimidinic endonuclease 1) activity 8-fold. 5-hydroxymethyl-deoxyuridine (5-hmdU) is an important oxidation product of thymidine which is removed by single-strand selective monofunctional DNA glycosylase (SMUG1). Biochemical experiments with purified proteins indicated that UV-DDB stimulates the excision activity of SMUG1 on several substrates by 4-5-fold. Electrophoretic mobility shift assays indicated that UV-DDB displaced SMUG1 from abasic site products. Single-molecule analysis revealed that UV-DDB decreases the half-life of SMUG1 on DNA by more than 8-fold. Immunofluorescence experiments demonstrated that cellular treatment with 5-hmdU (5 µM for 15 min), which is incorporated into DNA during replication, produces discrete foci of DDB2-mCherry, which co-localize with SMUG1-GFP. Proximity ligation assays supported a transient interaction between SMUG1 and DDB2 in cells. Poly (ADP)-ribose accumulated after 5-hmdU treatment, which was abrogated with SMUG1 and DDB2 knockdown. Moreover, we observed changes in cell bioenergetics during active 5-hmdU repair. These data support a novel role for UV-DDB in the processing of the oxidized base, 5-hmdU
Utilizing oncolytic vaccinia virus to modulate the tumor microenvironment
No full textOncolytic viruses are viruses which lyse tumor cells and stimulate a patient specific anti-tumor and anti-viral immune response. While one oncolytic virus has received FDA approval, clinical success has been limited. To design better oncolytics, we must improve our understanding of their mechanism of action and resistance. In this dissertation, I report that the infection of lymphocytes in the tumor by oncolytic vaccinia virus (VV) is important for the anti-tumor immune response. VV infects suppressive exhausted T cells and leads to their death and inhibiting this death decreases animal survival. I also found that oncolytic vaccinia-resistant tumors have high levels of intratumoral TGFβ. By engineering oncolytic vaccinia to deliver a TGFβR inhibitor, we rendered these tumors sensitive to treatment. Inhibiting TGFβ in the tumor with the immune stimulatory effects of VV reduced the suppression of regulatory T cells, resulting in increased effector T cell function. Overall, we show that oncolytic viruses stimulate an inflammatory immune response in both sensitive and resistant tumor models. In tumors resistant to therapy, it is also necessary to target the suppressive cell types for deletion or reprogramming, to promote a response to VV therapy. While traditionally, oncolytic virus combinations have added more stimulatory therapies, these data suggest the importance of also targeting resistance mechanisms when engineering or combining oncolytics with other therapies
Bacteriophage-host interactions in Mycobacterium and related actinobacteria
No full textEvery year, Mycobacterium tuberculosis (Mtb), the causative agent of the disease tuberculosis kills nearly two million people globally. In recent times this public health emergency has been exacerbated by acquired antibiotic resistance. Even though this was one of the first etiologic agents of disease to be discovered, many aspects of the physiology of Mtb remain unknown. Mycobacteriophages, viruses that can infect Mtb and closely related bacteria may be useful tools to both combat and study Mtb. In this work, I address three aspects relating to the dynamics between phages that infect Mtb and related bacteria in the phylum Actinobacteria.
First, the therapeutic potential of mycobacteriophages is explored by elucidating the susceptibility to phage infection for a set of strains of Mtb that is representative of the global genetic diversity of this pathogen. This investigation led to propose a five-phage cocktail that is able to both infect and kill representative strains of Mtb.
Secondly, as resistance occurs with antibiotics, phage resistance can also arise. Phage resistance can occur through spontaneous mutations that alter host-factors that are required for phages to successfully infect their host. In Mycobacterium few of the host-factors associated with phage resistance have been elucidated. Through the work presented here, several host-factors associated with phage resistance in Mycobacterium are presented and characterized, including the nucleoid associated protein Lsr2 in M. smegmatis, as well as genes of the methylglucose lipopolysaccharide (MGLP) pathway that cause phage resistance in both M. smegmatis and Mtb.
Finally, I describe tRNA-dependent lysogeny, an unusual and previously uncharacterized lysogenization mechanism that depends on a phage-encoded tRNA. Phages that encode tyrosine integrases frequently integrate into tRNA genes in their host and reconstitute the 3’ end of the tRNA gene upon integration. Phages that employ tRNA-dependent lysogeny do not reconstitute the tRNA, and instead use a tRNA present on their own genome to complement the loss of the host tRNA. This new strategy of lysogeny is employed by phages that infect the Actinobacterial hosts Mycobacterium and Gordonia and in many ways is counter to the model for integration and lysogeny that was established in E. coli phage Lambda
The Role of Protein Kinase D2 in Ischemic Stroke
No full textIschemic stroke, constituting 80–90% of all strokes, is a leading cause of death and long-term disability in adults. There is an urgent need to discover new targets and therapies for this devastating condition. Protein kinase D (PKD), as a key target of diacylglycerol involved in ischemic responses, has not been well studied in ischemic stroke, particularly PKD2. In this study, we found that PKD2 expression and activity were significantly upregulated in the ipsilateral side of the brain after transient focal cerebral ischemia, which coincides with the upregulation of PKD2 in primary neurons in response to in vitro ischemia, implying a potential role of PKD2 in neuronal survival in ischemic stroke. Using kinase-dead PKD2 knock-in (PKD2-KI) mice, we examined whether loss of PKD2 activity affected stroke outcomes in mice subjected to 1 h of transient middle cerebral artery occlusion (tMCAO) and 24 h of reperfusion. Our data demonstrated that PKD2-KI mice exhibited larger infarction volumes and worsened neurological scores, indicative of increased brain injury, as compared to the wild-type (WT) mice, confirming a neuroprotective role of PKD2 in ischemia/reperfusion (I/R) injury. Mouse primary neurons obtained from PKD2-KI mice also exhibited increased cell death as compared to the WT neurons when subjected to in vitro ischemia. We have further identified AKT and CREB as two main signaling nodes through which PKD2 regulates neuronal survival during I/R injury. To determine the therapeutic potential of targeting PKD2 in ischemic stroke, we designed and evaluated PKD2-activating tat-peptides to determine whether targeted activation of PKD2 promotes neuronal survival and functional recovery. D2tatAD peptide specifically, increased neuronal viability and proliferation during I/R injury and reduced infarction size in mice after tMCAO along with increased activation of PKD and downstream AKT and CREB in line with the activation of the PKD2 neuroprotective pathway. Overall, our study demonstrated that PKD2 conferred neuroprotection in ischemic stroke by promoting AKT and CREB activation and targeted activation of PKD by a tat-peptide D2tatAD promoted neuronal survival in response to I/R injury in vitro and in vivo. Our findings highlight the therapeutic potential of targeting PKD for treatment of ischemic stroke where new therapies are in high demand
Neuroimmune Gene Expression in Alcohol Use Disorder
No full textAlcohol use disorder (AUD) is a debilitating psychiatric condition that impacts millions of people in the United States. Chronic alcohol exposure induces a number of molecular and biological changes, including increasing neuroinflammation and transcriptomic alterations. While much of the focus to date has been on protein coding genes when characterizing genetic changes associated with AUD, interest has begun to shift to long noncoding RNAs (lncRNAs). LncRNAs are now widely recognized as active molecules regulating processes from cell proliferation and neuroinflammation to behavior. With this knowledge, the work presented here aimed to characterize neuroinflammatory-related transcriptomic changes following ethanol exposure and begin to understand how these changes contribute to AUD. To begin, extracellular vesicles were isolated from ethanol exposed brains and the RNA content was examined. Due to their role in neuroinflammation, I next moved into primary astrocyte cultures and characterized transcriptomic changes following ethanol exposure in both males and females. Finally, I focused my interest in lncRNA on metastasis associated lung adenocarcinoma transcript 1 (Malat1), which is increased in the brain following ethanol exposure. Changes in the glial transcriptome, as well as alternative splicing, were characterized following Malat1 knockdown with and without ethanol exposure. Then, using both a global knockout mouse and a global, inducible knockout mouse line, the impact of Malat1 knockout was assessed on ethanol consumption and preference, among other ethanol-related behaviors such as tolerance and anxiety-like behavior. Overall, ethanol exposure induced significant and sex-dependent changes in the RNA content of extracellular vesicles and in primary astrocyte cultures, which corresponded with biological processes such as neuroinflammation, extracellular matrix organization, and cell proliferation. Malat1 knockout in glial cultures also revealed distinct sex-dependent mechanisms, wherein female cultures demonstrated significant changes in inflammatory responses not found in male cultures. Finally, Malat1 knockout in vivo led to female-specific decreases in ethanol consumption and preference in both mouse lines. Together, these findings highlight the transcriptome as an integral part of controlling the effects of chronic ethanol exposure and highlight the importance studying biological sex in the study of AUD. These studies also establish Malat1 as a key regulator of neuroinflammation and ethanol consumption in females
The Longitudinal Impact of Maladaptive Stress Response in Offspring with Parental Cancer
No full textEarly life stress (ELS) comprises a wide range of negative events—along with the resultant physiologic responses—that children experience during early childhood. While its prevalence has been well documented, mounting evidence suggests that the long-term consequences of stress responses to ELS can be profound both in their severity and longevity, with studies showing that adults who experienced high levels of ELS face increased risk for the development of not only multiple psychiatric diagnoses and behavioral disturbances, but also for more disparate diseases such as cancer, cardiovascular disease, and premature death, with all of the above risks persisting indefinitely into adulthood. While much remains unknown regarding the mechanisms underlying this psychopathology, the Hypothalamic-Pituitary-Adrenal (HPA) axis and inflammatory pathways have been implicated.
Many challenges face researchers assessing ELS, including barriers to reaching children soon after exposure, strong associations between differing sources of ELS, and issues regarding timing of stressor onset. Recently, parental cancer has been identified as a source of unanticipated and persistent stress that addresses many of these limitations; these offspring are accessible soon after their parent’s diagnosis, which is a discrete and known event in time. Moreover, there is evidence to suggest that unlike many forms of ELS, these offspring are not at increased risk for carrying prior psychiatric burden into the study—as cancer’s prevalence is fairly consistent across socioeconomic backgrounds—nevertheless following diagnosis they are placed at increased risk for developing clinical symptomatology.
This dissertation is the first analysis of a large longitudinal study following young offspring of parents with cancer soon after diagnosis. We assessed these offspring and compared them to controls across a wide array of clinical indices, including depressive symptoms and functional impairment as our primary outcomes, prior risk and protective factors, biological markers for inflammatory and HPA-axis activity, and gene expression markers underlying implicated pathways. In doing so, we aim to gain insight into the genetic, biologic, and clinical disturbances that are associated with a discrete and identifiable source of ELS, as well as the pathological processes that presage development of maladaptive responses and the eventual onset of symptomatology more generally