Doctor of Philosophy

dissertationSpinal cord injury is debilitating and patients have historically had little hope for functional recovery. Astrocytes forming the scar tissue and producing chondroitin sulfate proteoglycan (CSPG) were often identified as the chief culprits for inhibiting neuronal regeneration. However, as the complex role of astrocytes in spinal cord injury are increasingly understood, the benefits they provide for healing have become valuable tools towards promoting recovery. Inducing aligned astrocyte populations in the wound site may promote functional recovery outcomes. Many approaches have been researched for restoring signaling after spinal cord injury, but artificial nerve guidance devices are particularly interesting as they can provide lasting directional cues to the injury site. The goal of this research was to demonstrate and measure the impact of surface protein patterns on astrocyte reactivity in an effort to reduce their neuroninhibitory CSPG production. By quantifying CSPG expression as measure of astrocyte reactivity, no change in reactivity was seen with varying amounts of fibrinogen surface coverage. However, astrocytes were found to selectively remove adsorbed fibrinogen from glass surfaces from amongst other proteins and also secrete CSPG onto surfaces. To mitigate protein pattern removal, a cross-linked patterning method based on microcontact printing was developed to attach protein patterns to collagen gels. Protein patterns were first created on glass and transferred to collagen by gelling collagen directly on top of the pattern. This construct was then peeled off from glass to create free standing collagen sheets with one patterned surface. Stripe patterns of various extracellular matrix molecules were thus patterned and found to align astrocytes on collagen gel surfaces and reduced astrocyte CSPG expression. To understand how astrocytes interact with surface patterns, astrocyte morphology was measured in real time. This revealed that astrocytes prefer adhesion to laminin over aggrecan and will shift their cell bodies accordingly. Astrocytes initially extend multiple processes upon attachment and span the largest distances when presented with mixed adhesion cues, up to 150 micrometers. Collectively, this research demonstrates the importance of surface protein patterns in biasing astrocyte behavior and provides a new technique for further modifications of collagen hydrogels for use as nerve guidance materials

Single-step flask to 250 L cell culture with a hybrid mixing single-use bioreactor

The process of scaling suspended cell cultures from frozen stocks through a large single-use bioreactor (SUB) includes numerous processing steps and operations. These steps, or seed train, involve vessel transfers and associated connections that increase the potential for contamination and human error. Of the multiple methods available for inoculating a 250 L SUB with sufficient cell quanitites, including multiple shake flasks, tabletop stirred tank bioreactor, or a 50L SUBs. Over the past decade tilting-type single-use bioreactor has been particularly attractive. Such wave-inducing systems implement tilting or rocking motion to agitate cultures which provides gas transfer and maintains cell suspension. Additionally, these rocking systems capitalize on single-use bioprocess containers (BPCs) which facilitate rapid deployment and cleanup when compared to reusable alternatives. Unfortunately, while rocking systems are simpler than other options, they still require connection and transfer steps. In an effort to further streamline the scale-up process, a tilting mechanism and bottom heating jacket have been added to a standard 250 L SUB to provide rocking motion and bottom temperature control to the BPC. This hybrid system can thus utilize rocking motion to mix small volumes of liquid (10-20L) and maintains standard impeller-based stirring functionality for volumes around 50 L and above. As designed, proliferating cells in 500 mL of media from flasks could be directly added to small volumes of media in the tilting SUB. Additional expansions with media addition are supported with rocking and bottom heating. An example expansion sequence would take cells in 500 mL media in a flask to 1.5 L of fresh media to form 2 L in the hybrid SUB. This would be followed by 8 L of fresh media addition after sufficient growth to form 10 L of media. 40 L of fresh media could then be added after cell growth and once media volume has reached 50 L. At this point, mixing via rocking will be discontinued and operation would follow well defined 5:1 turndown settings using a standard pitched blade impeller and drilled hole sparger (DHS) for the remaining SUB culture process. Currently, overhead gassing coupled with liquid rocking is used primarily for expanding cultures. As low cell density shake flask and scale-up process do not often feature active feedback control, future cell-specific processing could be optimized as the 250 L SUB already equipped with integrated monitoring and controls for gassing, mixing, pH, dO2, dCO2, cell mass, and/or other emerging process analytical technologies (PAT). Implementation of such a hybrid SUB would reduce expenses, manual operations, and potential risk of process contamination. The 250L working volume SUB cultured to high density is sufficient for seeding multiple 1000L or 2000L SUBs in a production environment using a 5:1 turn-down. Alternatively, this concept should be of particular interest to those possessing high productivity cell lines that have need to generate clinical grade and quantities of materials, while only investing in only one piece of bioreactor equipment or they desire a scale-up solution that optimizes limited facility work spac

Continuous process performance enhancements for 50 L to 500 L single-use bioreactors: A technical comparison of erformance characterization, cell culture, and scale-up modeling

Improvements in single-use systems have allowed implementation of high-density cultures in emerging bioprocess work flows. Specifically, advantages of single-use bioreactors have been realized in perfusion applications in high-density seed train intensification or as a compact production-scale bioreactor system. Due to this and additional progressive advances in media optimization and improved clone genetic selection have increased stress on the perceived performance limitations of single-use bioreactors. This study shows integration of the Thermo Scientific™ HyPerforma™ Single-Use Bioreactor (S.U.B.) and how strategic enhancements to the sparger and agitation systems have revealed the potential for 3-4X improvement of mixing and mass transfer performance compared to legacy SUB designs. This work includes: 1) Bioreactor characterization and scalability analysis of the S.U.B. when targeting perfusion applications from 50 L pilot scale to 500 L production scale working volumes. 2) High-density culture results (\u3e200E06 cells/mL) while maintaining proper operating parameters using a TruBioTM DeltaV controller and online process analytics. New data reveals specifically how a 50 L S.U.B. equipped with a specialized precision drilled hole sparger (DHS), single use foam probe, and oversized impeller is able to improve overall SUB operating efficiency. Coupled with best practices and the desirable process benefits achieved through automation and control of vital process parameters, evidence is provided as to the advantages of continuous processing in single-use systems

Optimization of the single use bioreactor for growth and bead-to-bead transfer of Vero cells cultured on microcarriers

Scale up and vaccine production processes of adherent cells, such as Vero cells face many challenges. The fundamental steps of equipment selection and chosen operating parameters have a significant impact upon the detachment and reattachment of cells through the scale up process. Microcarriers greatly increase the surface area for adherent cells and offer flexibility for expansion to bioreactors, but scale-up methods require optimization of the mixing within the vessel and also optimization of how the cells are transferred from bead to bead at each step in the seed train. In this study we take a process previously shown to a work in spinner flasks (\u3c1L)1 and demonstrate how the 50L Thermo Scientific™ HyPerforma™ Single-Use Bioreactors (S.U.B.) can be optimized for growing and scaling adherent cells on microcarriers, methods for bead-to-bead transfer of the cells at each scaling step, and final cell isolation using the Harvestainer single use bead capture bag. References Hachmann A, Campbell A, Gorfien S. Scale-up Optimization of Vero Cells Cultured on Microcarriers in Serum-Free Medium for Vaccine Production. Poster presented at: Vaccine Technology VI; 2016 June 12-17; Albufeira, Portugal

Improved DynaBead removal using designed-for-purpose BioProcess containers

The recent FDA approval of a CAR-T cell based therapy has been an important milestone for progress in how we collectively can target and treat diseases. A key tool in these therapies have been Dynabeads for selection and activation of target cells. One important workflow step is the removal of Dynabeds after their use and prior to patient administration of cells. To further improve Dynabead removal outcomes for users of the DynaMag CTS system, we have endeavored to create a bioprocess container (BPC) with improved Dynabead retention characteristics. This bag leverages existing film and manufacturing expertise from Thermo Fisher Scientific that have been extensively used in the biopharmaceutical industry over the past decade. Fundamentally, this designed-for-purpose BPC increases the flow path of fluid over the DynaMag to provide longer exposure to magnetic fields. This in turn provides for superior Dynabead removal from the fluid when compared to standard bags without this design as demonstrated in Figure 1. Please click Additional Files below to see the full abstract

A surrogate-based approach for post-genomic partner identification

BACKGROUND: Modern drug discovery is concerned with identification and validation of novel protein targets from among the 30,000 genes or more postulated to be present in the human genome. While protein-protein interactions may be central to many disease indications, it has been difficult to identify new chemical entities capable of regulating these interactions as either agonists or antagonists. RESULTS: In this paper, we show that peptide complements (or surrogates) derived from highly diverse random phage display libraries can be used for the identification of the expected natural biological partners for protein and non-protein targets. Our examples include surrogates isolated against both an extracellular secreted protein (TNFβ) and intracellular disease related mRNAs. In each case, surrogates binding to these targets were obtained and found to contain partner information embedded in their amino acid sequences. Furthermore, this information was able to identify the correct biological partners from large human genome databases by rapid and integrated computer based searches. CONCLUSIONS: Modified versions of these surrogates should provide agents capable of modifying the activity of these targets and enable one to study their involvement in specific biological processes as a means of target validation for downstream drug discovery

A specific group of genes respond to cold dehydration stress in cut Alstroemeria flowers whereas ambient dehydration stress accelerates developmental senescence expression patterns

Petal development and senescence entails a normally irreversible process. It starts with petal expansion and pigment production, and ends with nutrient remobilization and ultimately cell death. In many species this is accompanied by petal abscission. Post-harvest stress is an important factor in limiting petal longevity in cut flowers and accelerates some of the processes of senescence such as petal wilting and abscission. However, some of the effects of moderate stress in young flowers are reversible with appropriate treatments. Transcriptomic studies have shown that distinct gene sets are expressed during petal development and senescence. Despite this, the overlap in gene expression between developmental and stress-induced senescence in petals has not been fully investigated in any species. Here a custom-made cDNA microarray from Alstroemeria petals was used to investigate the overlap in gene expression between developmental changes (bud to first sign of senescence) and typical post-harvest stress treatments. Young flowers were stressed by cold or ambient temperatures without water followed by a recovery and rehydration period. Stressed flowers were still at the bud stage after stress treatments. Microarray analysis showed that ambient dehydration stress accelerates many of the changes in gene expression patterns that would normally occur during developmental senescence. However, a higher proportion of gene expression changes in response to cold stress were specific to this stimulus and not senescence related. The expression of 21 transcription factors was characterized, showing that overlapping sets of regulatory genes are activated during developmental senescence and by different stresses

Kinome-Wide siRNA Screening Identifies Src-Enhanced Resistance of Chemotherapeutic Drugs in Triple-Negative Breast Cancer Cells

Background: Chemotherapy is the main treatment for triple-negative breast cancer (TNBC), which lack molecular markers for diagnosis and therapy. Cancer cells activate chemoresistant pathways and lead to therapeutic failure for patients with TNBC. Several kinases have been identified as chemoresistant genes. However, the involvement of kinases in the chemoresistance in TNBC cells is not fully understood.Methods: We employed a kinome siRNA library to screen whether targeting any kinases could increase the chemosensitivity of TNBC cell lines. The effects of kinase on cell viability in various breast cancer cells were validated with ATP level and colony formation. Protein expression and phosphorylation were determined by immunoblotting. The Cancer Genome Atlas (TCGA) dataset was collected to analyze the correlation of Src expression with prognosis of TNBC patients.Results: Primary screening and validation for the initial hits showed that Src kinase was a potential doxorubicin-resistant kinase in the TNBC cell lines MDA-MB-231 and Hs578T. Both siRNA against Src and the Src inhibitor dasatinib enhanced the cytotoxic effects of doxorubicin in TNBC cells. Moreover, phosphorylation of AKT and signal transducer and activator of transcription 3 (STAT3), downstream effectors of Src, were accordingly decreased in Src-silenced or -inhibited TNBC cells. Additionally, TCGA data analysis indicated that Src expression levels in tumor tissues were higher than those in tumor-adjacent normal tissues in patients with TNBC. High co-expression level of Src and STAT3 was also significantly correlated with poor prognosis in patients.Conclusion: Our results showed that Src-STAT3 axis might be involved in chemoresistance of TNBC cells

Inflammatory myofibroblastic pseudotumour of the liver in association with gall stones - a rare case report and brief review

Inflammatory myofibroblastic pseudotumours of the liver are rare tumour-like lesions that can mimic malignant liver neoplasms. The symptoms and radiological findings of this rare tumour can pose diagnostic difficulties. We describe a 69-year-old gentleman who was admitted to our department with symptoms suggestive of acute cholecystitis. Ultrasonography and computed tomography of the liver raised the possibility of metastatic liver disease. A core biopsy of the liver was performed to confirm the diagnosis of liver metastasis. Unexpectedly it showed no evidence of malignancy but instead revealed an inflammatory myofibroblastic pseudotumour of the liver. This case report highlights the diagnostic dilemma that arose due to the similarity of appearances between the two pathological entities on imaging and this stresses the need for accurate histological diagnosis so as to avoid unnecessary surgical intervention. To the best of our knowledge, only a minority of cases are reported in the literature associating a hepatic inflammatory myofibroblastic pseudotumour with gall stones

Beneficial Effect of Consecutive Screening Mammography Examinations on Mortality from Breast Cancer: A Prospective Study

BackgroundPreviously, the risk of death from breast cancer was analyzed for women participating versus those not participating in the last screening examination before breast cancer diagnosis. Consecutive attendance patterns may further refine estimates.PurposeTo estimate the effect of participation in successive mammographic screening examinations on breast cancer mortality.Materials and MethodsParticipation data for Swedish women eligible for screening mammography in nine counties from 1992 to 2016 were linked with data from registries and regional cancer centers for breast cancer diagnosis, cause, and date of death (Uppsala University ethics committee registration number: 2017/147). Incidence-based breast cancer mortality was calculated by whether the women had participated in the most recent screening examination prior to diagnosis only (intermittent participants), the penultimate screening examination only (lapsed participants), both examinations (serial participants), or neither examination (serial nonparticipants). Rates were analyzed with Poisson regression. We also analyzed incidence of breast cancers proving fatal within 10 years.ResultsData were available for a total average population of 549 091 women (average age, 58.9 years ± 6.7 [standard deviation]). The numbers of participants in the four groups were as follows: serial participants, 392 135; intermittent participants, 41 746; lapsed participants, 30 945; and serial nonparticipants, 84 265. Serial participants had a 49% lower risk of breast cancer mortality (relative risk [RR], 0.51; 95% CI: 0.48, 0.55; P P ConclusionWomen participating in the last two breast cancer screening examinations prior to breast cancer diagnosis had the largest reduction in breast cancer death. Missing either one of the last two examinations conferred a significantly higher risk.Published under a CC BY 4.0 license.</p