Tyrosine Kinase Inhibitors associated muscle complaints like cramps, pain, and weakness are a few of the concerns contributing to declined disease control and quality of life. In this study, we investigated whether Ponatinib, a 3rd generation TKI, induces muscle toxicity in both In Vitro and In Vivo models and expand BMP-7 as a possible treatment option for its attenuation. For the In vitro study, Sol-8, a mouse myogenic cell line was exposed to Ponatinib and BMP-7 for 24hrs each. For the In vivo study, C57BL/6J mice were injected and euthanized after 14 days. The soleus muscle was isolated and used for experimentation. Both the studies were conducted with 3 groups: Control, Ponatinib, and Ponatinib+BMP-7. Ponatinib-induced muscle toxicity via apoptosis was established by TUNEL Assay, pro-apoptotic markers- Caspase 3, BAX, and anti-apoptotic marker Bcl2 via Immunocytochemistry and Immunohistochemistry. For the In vivo study, further confirmations of the above-mentioned apoptotic markers were done by RT-PCR. Ponatinib-induced muscle myopathy and loss in muscle function were also determined along with the effect on apoptotic pathway proteins PTEN and AKT. A significant (p < 0.05) increase in apoptotic positive nuclei as well as positive cells for pro-apoptotic markers was observed in Ponatinib treatment groups both in vitro and in vivo along with loss of muscle function and adverse muscle remodeling. Whereas BMP-7 treatment significantly (p < 0.05) attenuated Ponatinib-induced apoptosis, restored muscle function, and improved muscle remodeling. The result of our study suggests that Ponatinib-induces apoptotic cell death in skeletal cells which was attenuated by BMP-7.

Ames dwarf mice (df/df) display delayed aging relative to their normal siblings, living approximately 40-60% longer. As such, investigating the mechanisms that enable these organisms to have superior life expectancies may prove useful in developing therapies to slow aging and deter age-related disease. Nonalcoholic fatty liver disease (NAFLD) is a condition that is characterized by the accumulation of excess adipose tissue in the liver. Interestingly, its prevalence tends to increase with age with an incidence rate of greater than 40% in individuals over the age of 60. Previous studies highlight the potential of calorie restriction (CR) in promoting longevity, but little is known about its effects on the biomolecular processes that govern NAFLD. In this study, we explored the livers of Ames dwarf mice following 6 months of continuous calorie-restriction and discovered significant down regulation of candidate miR-34a, an established biomarker for fatty liver. Using qPCR and western blot, we found miR-34a to be correlated with the expression of age-related and lipid trafficking mRNA and protein. Specifically, CR upregulated Sirt1 in the Ames dwarf liver and subsequently induced gluconeogenesis and lipid catabolism. To verify the role of miR-34a in influencing fatty acid metabolism, we transfected the human liver cancer (HepG2) cell line with miR-34a mimic and observed its effect on direct targets SIRT1, AMPKa, and PPARa which are believed to influence downstream lipid transport genes. In all, our findings suggest that CR is a robust driver of the SIRT1 signaling pathway which reverses the pathology associated with age-related diseases by influencing insulin signaling in the Ames dwarf liver thereby improving fatty acid oxidation and cholesterol efflux and maintaining lipid turnover via autophagy.

Doxorubicin (Dox) is a potent and widely used anthracycline for cancer chemotherapy. However, the prevalence of Dox induced cardiotoxicity (DIC) in children and adolescents receiving Dox treatment is a major concern. While the exact molecular mechanisms of DIC remains to be fully elucidated, studies have reported cell death (apoptosis) as a major molecular mechanism in the occurrence of DIC. However, the presence of necroptosis in young DIC remains unknown. Therefore, in this study, we investigated the possible occurrence of necroptotic cell death in DIC in young mice in-vivo to delineate underlying pathological mechanism and the therapeutic ability of proprotein convertase substilisin/Kexin 9 inhibitors (PCSK9i) to attenuate doxorubicin induced necroptotic cell death and adverse cardiac remodeling. C57BL6 mice (6 + 2 weeks of age) were divided into three groups and administered the following treatments: Control (saline), Dox and Dox+PCSK9i. At D14 echocardiography was performed to examine heart function. Heart tissues were collected and analyzed for protein expression using immunohistochemistry and western blot to determine necroptosis and necroinflammation. Adverse cardiac remodeling was determined with histology stains for fibrosis and cardiac hypertrophy. Dox treatment significantly (p < 0.05) increased the expression of necroptotic markers [Receptor interacting Serine/Threonine kinase 1 and 3 (RIPK1, RIPK3) and Mixed Lineage kinase domain-Like (MLKL)], Necroinflammation markers [interleukin-33 (IL33) and interleukin-1α (IL-1α)], pro-inflammatory cytokines [interleukin- 6 (IL-6) and tumor necrosis factor alpha (TNF-α)], and induced adverse cardiac remodeling with significant increase (p < 0.05) in cardiac hypertrophy and fibrosis and significantly (p < 0.05) reduced cardiac function when compared to control. However, PCSK9i treatment showed a reduction in expression of necroptotic markers, necroinflammation markers and pro-inflammatory cytokines IL-6 and TNF-α, significant (p < 0.05) reductions in cardiac hypertrophy and fibrosis with improved heart function. Lastly, the result of our study shows that PCSK9i attenuates doxorubicin induced necroptosis and cardiac remodeling

Peripheral nerve injuries (PNI) affect 20 million people, with over 200,00 nerve repairs performed annually in the United States alone. Nerve conduits (NCs) are a commonly employed treatment for injured nerve segments under 3 cm, but unsatisfactory outcomes have been reported for larger gaps. This is in part due to the lack of bioactive agents such as stem cells or growth factors incorporated into FDA-approved NCs. While they have been experimentally shown to promote peripheral nerve regeneration (PNR), their inherent variability, harvesting complexity, and sometimes inadvertently high biological activity pose great regulatory hurdles. Therefore, plant extracts have become an attractive alternative area of bioactive agents proven to improve PNR while overcoming these issues, one of which is curcumin. However, studies largely use unoptimized curcumin which presents an issue as it is hydrophobic and degrades quickly in aqueous solutions. In this study, a nano-formulation comprised of tannic acid and polyvinylpyrrolidone (TA-PVP) was developed to encapsulate curcumin and optimize its delivery to cells. Results show that the developed Cur-TA-PVP nanoparticles (NPs) had an average size of 220 nm, a surface charge of -35mV, and a polydispersity index of 0.3. Further, curcumin was shown to be localized within the core of the NP. This significantly increased the colloidal stability, decreased the degradation rate, and promoted the slow release of curcumin in aqueous solutions. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the Cur-TA-PVP NPs was also shown to be superior as compared to free curcumin. Finally, fluorescence microscopy revealed a significantly higher concentration of curcumin in SH-SY5Y, S16 rat Schwann cells, and J774 murine macrophages when delivered via NPs. These results collectively provide evidence for the improved physicochemical properties of the nano-curcumin, which led to improved delivery to cells. Therefore, this formulation has the necessary attributes for incorporation into NCs to promote functional recovery following PNI.

Staphylococcus aureus nasal carriage (SANC) is largely asymptomatic, but presents a risk of autoinfection and dissemination to new immunocompromised hosts. SA disease states range from mild cutaneous infections to life-threatening bacteremia. Historically utilized rodent models do not naturally carry SA in the nose, are insufficient in longitudinal SANC experimentation, and lack immune factors that are vital in human clearance of SA. The nasal passages of non-human primates are similar anatomically and histologically, and reproductive mucosal studies have shown similar immune responses to pathogens and human-relevant microbial profiles. Seventeen captive pigtailed macaques (Macaca nemestrina) were found to naturally carry SA in the nose and pharynx, while topical mupirocin ointment effectively decolonized SA, similar to humans. Colonization was established with a human-relevant inoculum of 104 SA CFUs per nostril in four independent experiments, including with a human isolate (ST398). Autologous and non-autologous macaque strains were carried similarly in load and duration, each surviving over 40 days. Animals that cleared SA showed a rapid neutrophilic innate response, with up-regulation of IL-8, MCP-1, and IL-1β following inoculation, as observed in human hosts. Assessment of the nasal microbiome of pigtailed macaques and humans demonstrated similar relative abundance of the most prevalent genera: Staphylococcus, Corynebacterium, and Acinetobacter. Collectively, these multidimensional analyses provide evidence that the pigtailed macaque is a novel physiological model of human SANC that may be useful for testing novel SA decolonization strategies.

The brewing industry generates $350 billion in revenue in the US annually, representing 1.9% of the gross domestic product. Spoilage is a persistent problem throughout production and distribution that causes economic loss, and is therefore meticulously avoided. Contrarily, artisanal sour beers are necessarily produced by a diverse variety of these spoilage organisms metabolically interacting in symbiosis as a microbial ecosystem. We sought to gain insight into factors driving assembly of microbial communities by testing a long-debated Darwinian hypothesis. A collection of community members were screened in co-culture and novel bioinformatics tools were developed to predict observed interactions. A fundamental understanding of these relationships is paramount to beer production and sets a precedent for the study of similar microbial communities that impact human health.

In Escherichia coli, growth is rate-limited by translation capacity [1]. Stalled ribosomes have profound effects on a cell such as altered mRNA abundance, decreased ribosome availability, and an imbalanced proteome. The absence of elongation factor P (EF-P), a universally conserved transpeptidation enhancer, presents an extreme example of this scenario, wherein ribosomes accumulate disproportionately onto messages that are more slowly translated and cell growth becomes notably impaired. We discovered that faster-growing cells arise spontaneously in Δefp cultures, suggesting that translation defects could be circumvented by mutating other genes. This thesis presents a genetic and biochemical analysis of a mechanism Δefp cells employ to overcome translation stress. Using a dual luciferase reporter system, we found that transpeptidation remained hindered in the faster growing Δefp cells. Whole genome sequencing of several fast-growing strains revealed mutations in a poorly characterized RNA helicase called HrpA. We determined that deletion of hrpA, or mutations at several conserved residues critical for HrpA's function, was sufficient to improve the fitness of Δefp cells. HrpA is a DEAH-box RNA helicase and represents a large class of enigmatic proteins that use ATP to restructure cellular RNAs; however, it's direct function in cellular physiology has yet to be clearly demonstrated [2, 3]. Several HrpA mutants were engineered to interrogate the molecular mechanism of HrpA and how its function impairs Δefp cells. Complementation in Δefp ΔhrpA cells showed that a number of these mutants were unable to restore sickness, suggesting they were defective in key aspects of RNA processing. It was discovered that wild-type HrpA is associated with actively translating ribosomes and several of the inactive HrpA mutants impose substantial deleterious effects on translation and ribosome production. In sum, the work presented here describes a mechanism by which cells overcome translation stress involving a novel genetic and biochemical relationship between EF-P and HrpA.

Litteraturstudier har genomförts med syftet att utveckla minst en ny detektionsmetod som skulle kunna ersätta den metod som företaget Cavidi använder sig av idag. Cavidi hade specificerat krav som metodförslaget skulle uppfylla. Dessa krav var att metoden skulle vara snabb, lättanvänd, billig, ha hög känslighet och kunna förvaras i rumstemperatur. Två lovande metoder som baseras på två olika principer valdes ut. Den ena metoden bygger på att enkelsträngat DNA med en specifik nukleinsyrakomposition syntetiseras. Denna sekvens har egenskapen att spontant bilda en sekundärstruktur som kan bilda komplex med en fluorofor. Detta ökar dess fluorescens. Ökningen detekteras med fluorescensspektroskopi. Den andra metoden baseras på ett optomagnetiskt fenomen vilket innebär att ett magnetiskt fält påverkas av polariserat ljus. Metoden går ut på att virioner först renas fram från blodplasma och att de fäster på jonbytarkulor under rådande buffertförhållanden. Magnetiska nanopartiklar tillsätts som binder till jonbytarkulornas lediga ytor. Om många virioner har bundit till jonbytarkulorna finns det en större mängd fria nanopartiklar i lösningen. Antalet fria nanopartiklar i lösningen är proportionellt mot mängden HIV i provet och kan då detekteras med en fotodetektor. Fördelarna med dessa metoder är att processen blir billigare, snabbare och har en hög känslighet. Metoderna är lättanvända och använder färre komponenter jämfört med Cavidis nuvarande metod. Därmed blir Cavidis produkter billigare och tillgängliga för fler människor.

In the human body, the main function of the healthy thyroid gland is the regulation of the metabolism and hormone production. Included in the thyroid are organized structured and uniformly shaped follicles ranging from 50-500 μm in diameter. Pathologies lead to morphological changes of these follicles, affecting the density and size, but can also lead to an absence. In this study optical coherence tomography (OCT) was used to examine pathological thyroid tissue by extracting structural information of the follicles from image segmentation. However, OCT images usually include a high amount of speckle noise which affects the segmentation outcome. Due to that, the OCT images need to be improved. The aim of this thesis was to investigate the appropriate filtering methods to enhance the images and thus improve the segmentation outcome. The images of pathological thyroid tissues with a size of 0:5-1 cm where scanned by a spectral domain OCT system (Telesto II, Thorlabs GmbH, Germany) using a center wavelength of 1300nm. The obtained 2D and 3D images were saved as .oct file as well as implemented and visualized in a MATLAB graphical user interface (GUI) for further processing. For image improvement, four filtering enhancement methods were applied to the 2D images such as the enhanced resolution imaging (ERI), adaptive Wiener filter, discrete wavelet transform (DWT) and multi-frame wavelet transform (WT). The processed images were further converted to grayscale and binary images for intensity-based segmentation. The output of all methods were compared and evaluated using signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), enhanced number of looks (ENL), edge profiles and outcome of the segmented images. It was demonstrated that the complex DWT (cDWT) with a higher threshold and the multi-frame WT using the haar wavelet showed enhanced results over the other filtering methods. The computed SNR could be increased up to 52% and the ENL value up to 4802%, applying the multi-frame WT, while the CNR could be increased up to 106% for cDWT. The lowest obtained gradient was equal to an intensity decrease of -61% and -68% for multi-frame WT and cDWT, respectively. The filtering method could increase the smoothness of the image while the edge sharpness could be kept. The segmentation could detect both small and large follicles. ERI did not show any improvement in the segmentation but could enhance the structural detail of the image. Larger neighbourhoods of the adaptive Wiener filter showed a highly blurred image and led to merged follicles in the image segmentation. The wavelet filters DWT and multi-frame WT gave most satisfying results since high and low frequencies were divided into subbands, where individual information on vertical, horizontal and diagonal edges was stored. Applied cDWT had an even higher amount of subbands, so that more information on signal and speckle noise could be specified. Due to this fact, it was possible to achieve a decreased noise level while edge sharpness where maintained. Using a multi-frame image an increased SNR was obtained, as the intensity information stayed constant over the individual frames while the noise information changed. Wavelet based filtering showed higher improved results in comparison to the adaptive Wiener filter or the ERI in the 2D domain. By applying filtering methods in higher dimensions such as 3D or even 4D, better results in noise reduction are expected. Improved settings for the individual filtering methods as well as enhancement in segmentation are part of the future work.

digitalisering@umu.se