The question of whether within chimera research certain human/nonhuman animal chimeras should not be created has received significant attention in the philosophical community. In this thesis I examine, from a philosophical perspective, four questions related to this topic. These are: (i) Can ‘human’ dignity be invoked as an argument against the creation of human/nonhuman animal chimeras? (ii) Is it morally permissible to kill human/great-ape chimeras in order to solve the human organ shortage crisis? (iii) Is there something inherently wrong with creating human/nonhuman animal chimeras capable of human gamete production and human pregnancy? (iv) Can classic animal ethics issues concerning animal welfare throw light on the issue of killing human/pig chimeras for their human organs. These four questions, in turn, fall within what have been regarded as the three main problematic cases within chimera ethics: (1) the creation of human/nonhuman animal chimeras that could have brains predominantly constituted by human brain cells; (2) the creation of human/nonhuman animal chimeras that could look human-like; (3) the creation of human/nonhuman animal chimeras capable of human gamete production and human pregnancy. In the introduction I do four things. First, I define what chimeras are and point out some problems with some of the definitions that have been proposed. Secondly, I briefly investigate if there is a sound principled objection against the intentional creation of all intraspecific and interspecific chimeras. Thirdly, I present in a detailed fashion the four questions that this thesis poses in response. Finally, I explain how the four papers that are the main body of the thesis form a coherent body of work. In the fifth section of this thesis I present the abstracts of four papers. In the sixth section I make a comprehensive exploration of the dignity-based arguments that have been advanced against the creation of human/nonhuman animal chimeras that could possess human-like mental capacities, or that could possess certain cognitive capacities that we would not be able to properly classify. In the seventh section I examine the morality of killing human/great ape chimeras for their human organs. This paper is a detailed response to Shaw et al.’s (2014) ‘Using Non-Human Primates to Benefit Humans: Research and Organ Transplantation’. The eight section covers two topics. First I discuss the moral problems related to the creation of mainly nonhuman human/nonhuman animal chimeras capable of human gamete production. Secondly, I discuss the moral problems that would be generated by the fact that a sentient nonperson human/nonhuman animal chimera, that is predominantly nonhuman, was pregnant with a human conceptus. In the ninth section I further examine the chimera welfare issues that have emerged from assessing the morality of using human/pig chimeras as human organ sources. In the conclusion of the thesis I present a summary of the main points I have explored, and proceed to present how my dissertation adds to the academic literature on chimera ethics. Finally, I present some areas for further research.

No description available.

Assembly of virus capsid protein (CP) into icosahedrally symmetric particles is an intriguing and elegant process. In most cases of virus assembly, a large number of identical protein subunits self-assemble to generate a shell that protects the viral genome. Studies on virus assembly have resulted in a new scientific technique that uses these proteinaceous shells as nano-particles for a variety of biological applications. The current thesis deals with understanding the factors that govern the assembly of the Sesbania mosaic virus (SeMV) and a pleomorphic virus, Tobacco streak virus (TSV). CP of SeMV, a T=3 plant virus, consists of a disordered N-terminal R-domain and an ordered S-domain. The importance of the R-domain in the assembly was probed by replacement with polypeptides such as the B-domain of Staphylococcus aureus protein A and polypeptides P10 and P8 of SeMV. These chimera assembled into T=3 or larger virus like particles (VLPs). Addition of divalent cations resulted in the formation of heterogeneous nucleoprotein complexes that disappeared upon treatment with EDTA/RNAse. One of the chimeras (N∆65-B) purified in a dimeric form by affinity chromatography assembled into T=1 VLPs during crystallization. The three dimensional structure of these VLPs showed that they were devoid of divalent ions and the B-domain was disordered. These studies demonstrate the importance of N-terminal residues, metal ions in virus assembly and robustness of the assembly process. Also, the B-domain was functional in N∆65-B VLPs, suggesting possible biotechnological applications. Tobacco streak virus (TSV) is a polymorphic virus and a major plant pathogen. TSV capsids encapsidate the tri-partite ss-RNA genome of the virus in three spheroidal particles of diameters 27, 30 and 33 nm, respectively. CPs of ilarviruses are also involved in genome activation. The labile nature of ilarviruses has posed difficulties in their structure determination. This thesis describes the first crystal structure of truncated TSV-CP. The core of TSV CP conforms to the canonical β-barrel jelly roll tertiary structure found in other viral coat proteins. Dimers of CP with swapped C-terminal arms (C-arm) were observed in the two crystal structures determined. The C-arm was found to be flexible and responsible for the polymorphic and pleomorphic nature of TSV capsids. Mutations in the hinge region of the C-arm that reduce the flexibility resulted in the formation of more uniform particles. TSV CP was also found to be structurally similar to that of Alfalfa mosaic virus (AMV) accounting for similar mechanism of genome activation in alfamo and ilar viruses.

<p>S. 1-80: sammanfattning, s. 81-159: 7 uppsatser</p> / digitalisering@umu

abstract: Prior to the first successful allogeneic organ transplantation in 1954, virtually every attempt at transplanting organs in humans had resulted in death, and understanding the role of the immune mechanisms that induced graft rejection served as one of the biggest obstacles impeding its success. While the eventual achievement of organ transplantation is touted as one of the most important success stories in modern medicine, there still remains a physiological need for immunosuppression in order to make organ transplantation work. One such solution in the field of experimental regenerative medicine is interspecies blastocyst complementation, a means of growing patient-specific human organs within animals. To address the progression of immune-related constraints on organ transplantation, the first part of this thesis contains a historical analysis tracing early transplant motivations and the events that led to the discoveries broadly related to tolerance, rejection, and compatibility. Despite the advancement of those concepts over time, this early history shows that immunosuppression was one of the earliest limiting barriers to successful organ transplantation, and remains one of the most significant technical challenges. Then, the second part of this thesis determines the extent at which interspecies blastocyst complementation could satisfy modern technical limitations of organ transplantation. Demonstrated in 2010, this process involves using human progenitor cells derived from induced pluripotent stem cells (iPSCs) to manipulate an animal blastocyst genetically modified to lack one or more functional genes responsible for the development of the intended organ. Instead of directly modulating the immune response, the use of iPSCs with interspecies blastocyst complementation could theoretically eliminate the need for immunosuppression entirely based on the establishment of tolerance and elimination of rejection, while also satisfying the logistical demands imposed by the national organ shortage. Although the technology will require some further refinement, it remains a promising solution to eliminate the requirement of immunosuppression after an organ transplant. / Dissertation/Thesis / Masters Thesis Biology 2020

<p>The ubiquitin-proteasome system is responsible for cellular protein recycling, and it is a crucial system to maintain proper protein balances in cells. Proteasome is the main component of the system, and the system is tightly related to multiple cellular processes. Malfunction of the proteasome could lead to various diseases including cancer, neurodegenerative diseases and autoimmune diseases. As a result, researchers have been developing small molecules to target the proteasome to regulate its function. Currently, three small molecules have been approved by FDA as proteasome inhibitors to treat hematological cancer multiple myeloma. However, these small molecules inhibit the same enzymatic subunit on the proteasome and drug resistance has been observed among patients administrating these proteasome inhibitors. To develop new small molecules to target the proteasome, we started to investigate the 19S regulatory particle of the proteasome. In this work, we presented a workflow of discovering a small molecule selective binder, TXS-8, to 19S regulatory particle subunit Rpn-6. We also developed a series of assays to investigate the impact of small molecule on proteasome activity. At last, we introduced the binding site study of TXS-8, development of TXS-8-based PROTAC and new proteasome probe development.</p> <p>We first developed a one-bead-one-compound (OBOC) library to screen with Rpn-6 to discover potential binders to Rpn-6. After careful evaluation and validation, TXS-8 was discovered as the best hit from the screening. Our covalent pull-down experiment with cell lysate later confirmed TXS-8 as a selective binder of Rpn-6 and proteomic analysis of the pulled down protein also validated Rpn-6 as the major target of TXS-8.</p> <p>We then investigated the impact of TXS-8 in Rpn-6 overexpressed cancer cells like Ramos B-cell and multiple myeloma. TXS-8 was four-fold more toxic in these cells comparing to our control HEK-293T cells. To understand the cause of cell death when dosed with TXS-8, we began to investigate the impact of TXS-8 on proteasome activity, but some preliminary results were inconsistent. By the same time, there is also lack of a general workflow to investigate the impact of small molecules on proteasome activity. Therefore, we developed a three-step process to illustrate the general workflow using TXS-8 as an example. We first knocked down Rpn-6 in HEK-293T cells and monitored proteasome activity changes with a cell permeable probe our lab developed. We then transfected HEK-293T cells with a full-length foreign protein and knocked down Rpn-6 in these cells. We later monitored the degradation of the foreign protein when dosed with TXS-8. In the last step, we monitored the proteasome activity changes in primary cell lines when dosed with TXS-8. From these three steps, we successfully demonstrated a general workflow to investigate if a small molecule can affect proteasome activity. We also concluded that TXS-8 was unable to affect proteasome activity at non-lethal concentration.</p> <p> To further investigate TXS-8 and provide guidance for future structural optimization to improve potency, we proposed two methods on investigating the general binding site of TXS-8 on Rpn-6 using cross-linking techniques that is currently ongoing. We also modified TXS-8 into proteolysis targeting chimeras (PROTACs) to investigate if TXS-8-based PROTAC can improve toxicity and selectively induce Rpn-6 degradation in cells. However, no significant cell toxicity or Rpn-6 degradation was observed when dosed with TXS-8-based PROTACs.</p> Finally, Due to limitation of cell permeable probes, we were unable to investigate the impact of TXS-8 on the caspase-like β1 and trypsin-like β2 subunit of the proteasome in our previous studies. Although TXS-8 did not alter the chymotrypsin-like activity at non-lethal concentration, examining the effect of TXS-8 on the caspase-like and trypsin-like activity could still benefit our research. Besides, we also desire to expand our proteasome activity toolbox by developing more sensitive proteasome probes. Therefore, by analyzing and combing the commercially available proteasome probes and LLVY-Rh probes, we decided to develop selective proteasome probes for the β1 and β2 subunit to provide useful tools for future potential small molecule proteasome regulator characterization.

The genus Benyvirus includes the most important and widespread sugar beet viruses transmitted through the soil by the plasmodiophorid Polymyxa betae. In particular Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, causes an abnormal rootlet proliferation known as rhizomania. Beet soil-borne mosaic virus (BSBMV) is widely distributed in the United States and, up to date has not been reported in others countries. My PhD project aims to investigate molecular interactions between BNYVV and BSBMV and the mechanisms involved in the pathogenesis of these viruses.BNYVV full-length infectious cDNA clones were available as well as full-length cDNA clones of BSBMV RNA-1, -2, -3 and -4. Handling of these cDNA clones in order to produce in vitro infectious transcripts need sensitive and expensive steps, so Ideveloped agroclones of BNYVV and BSBMV RNAs, as well as viral replicons allowing the expression of different proteins.Chenopodium quinoa and Nicotiana benthamiana plants have been infected with in vitro transcripts and agroclones to investigate the interaction between BNYVV and BSBMV RNA-1 and -2 and the behavior of artificial viral chimeras. Simultaneously I characterized BSBMV p14 and demonstrated that it is a suppressor of posttranscriptional gene silencing sharing common features with BNYVV p14.

Conhecemos o papel dos leucócitos no controle do Trypanosoma cruzi, mas ignoramos qual a contribuição das populações estruturais (miócitos, hepatócitos, etc). Estas populações poderiam sinalizar a presença do parasita e/ou ter ação efetora que poderia aumentar em resposta a citocinas como o IFN-g. Para verificar se as células estruturais respondem ao IFN-g contribuindo à destruição do T. cruzi, analisamos a infecção em quimeras WT/IFNgR-KO (WT/KO) geradas em camundongos IFNgR-KO reconstituídos com medula óssea de camundongos selvagens (WT), nas quais parte dos leucócitos é IFNgR+, mas as células não leucocitárias são deficientes. Quimeras WT/KO e quimeras controle WT/WT foram estudadas em relação à carga parasitária e inflamação. O parasitismo sistêmico e tissular é maior nas quimeras WT/KO do que nas quimeras WT/WT, mas inferior à dos controles IFNgR-KO. Nos animais WT/KO o aumento de ninhos no coração e músculo não se acompanha de aumento no infiltrado leucocitário. Os resultados sugerem que as células não leucocitárias contribuem à defesa frente ao T. cruzi. / Although we know the role played by leukocytes in Trypanosoma cruzi control, we ignore the contribution of structural cells (myocytes, hepatocytes, etc). These cells could signal the presence of the parasite and/or mediate an effector activity which could increase in response to cytokines, as IFN-g. To evaluate if non-leukocytes respond to IFNg, contributing to T. cruzi destruction, we analysed the infection in WT/IFNgR-KO (WT/KO) chimaeras, generated in IFNgR-KO mice reconstituted with bone marrow of wild-type mice (WT) so that most leukocytes are IFNgR+ but structural cells are deficient. WT/KO chimaeras and control WT/WT chimaeras were infected by T. cruzi and the parasite load and inflammation analyzed in various organs. Systemic and tissue parasite loads were higher in WT/KO chimeras than in WT/WT chimeras, but lower than in control IFNgR-KO mice. In WT/KO chimaeras the increased parasite load at the heart and skeletal muscle was not followed by an increase of inflammatory infiltrates. Our results suggest that structural cells contribute to T. cruzi control.

Lectin nucleotide phosphohydrolases (LNPs) are proteins which possess both apyrase catalytic activity (E.C. 3.6.1.5) and specific carbohydrate binding properties, and these are linked. To investigate the structural and functional properties for these proteins, two putative soluble plant LNPs, 4WC and 7WC (from white clover), and a putative soluble plant apyrase 6RG (from ryegrass) were chosen. Rabbit polyclonal antibodies for each plant apyrase were generated using highly purified, overexpressed recombinant 4WC or 7WC. In the case of 6RG, the C-terminal half of the protein constituted the best antigen for generating polyclonal antibodies. These antibodies showed high specificity and sensitivity. Active, recombinant 4WC and 6RG were overexpressed and purified using the baculoviral insect cell expression system (4WCbac-sup and 6RG:Hisbac), while 7WC (7WCcoli) was produced from E. coli inclusion bodies and subsequently refolded to give active enzyme. In course of overexpression, recombinant 4WC was localised in both the cellular fraction (4WCbac) and in the media supernatant (4WCbac-sup), while recombinant 6RG:Hisbac was only found in the cellular fraction (6RG:Hisbac) indicating that it was not secreted during insect cell growth. Secretion of 4WCbac was found to be dependent on N-glycosylation at N313 but not at N85 and elimination of one or both of these sites appeared to have little influence on apyrase activity. In addition, both 4WCbac and 6RG:Hisbac from the cellular fraction were fully functional. These results were compared with similar work performed on the animal ecto-apyrases which have different specific N-glycosylation sites required for secretion and activity. The 4WCbac-sup, 7WCcoli and 6RG:Hisbac proteins all showed apyrase activity, that is they catalysed the hydrolysis of nucleotide tri- and/or di-phosphates to their corresponding nucleotide monophosphates, and released inorganic phosphate in a divalent cation-dependent manner. However, the proteins exhibited different activities, substrate specificities, pH profiles and influence of inhibitors: 4WCbac-sup had a preference for NDPs with a pH optimum ≥9.5; 7WCcoli had a modest preference for NTPs with a pH optimum at 8.5; 6RG:Hisbac was almost exclusively an NTPase with a pH optimum at 6.5. Contrary to predictions based on phylogeny the proteins all bound to sulphated disaccharides and their catalytic activities were influenced both positively and negatively by the binding of specific chitosans. The data indicates that all three soluble plant apyrases investigated here were LNPs, in contrast to predictions from the literature. In order to pinpoint the regions responsible for determining substrate specificity and chitosan binding, chimeras were made using the N- and C-terminal halves of 4WC and 6RG. This resulted in fully functional reciprocal chimeras. Comparison of the apyrase activity for parents and chimeras, substrate specificity, optimal pH, influence of inhibitors on activity and effects of chitosans indicated that the C-terminus was responsible for determining substrate specificity. However, the influence of specific chitosans on the chimeras appeared to be dependent on both the N- and C-terminal portions of the proteins. In addition, chimeras were found to bind to the same sulphated disaccharides as the parent proteins. Preliminary crystal screening experiments were performed with highly purified preparations of 7WCcoli and 6RG:Hisbac. Under specific conditions 7WCcoli was found to form cube-like crystalline arrangements while 6RG:Hisbac formed hexagonal-like crystalline structures. A potential model for carbohydrate binding by LNPs is proposed and the possible biological roles of plant LNPs are discussed.

Lectin nucleotide phosphohydrolases (LNPs) are proteins which possess both apyrase catalytic activity (E.C. 3.6.1.5) and specific carbohydrate binding properties, and these are linked. To investigate the structural and functional properties for these proteins, two putative soluble plant LNPs, 4WC and 7WC (from white clover), and a putative soluble plant apyrase 6RG (from ryegrass) were chosen. Rabbit polyclonal antibodies for each plant apyrase were generated using highly purified, overexpressed recombinant 4WC or 7WC. In the case of 6RG, the C-terminal half of the protein constituted the best antigen for generating polyclonal antibodies. These antibodies showed high specificity and sensitivity. Active, recombinant 4WC and 6RG were overexpressed and purified using the baculoviral insect cell expression system (4WCbac-sup and 6RG:Hisbac), while 7WC (7WCcoli) was produced from E. coli inclusion bodies and subsequently refolded to give active enzyme. In course of overexpression, recombinant 4WC was localised in both the cellular fraction (4WCbac) and in the media supernatant (4WCbac-sup), while recombinant 6RG:Hisbac was only found in the cellular fraction (6RG:Hisbac) indicating that it was not secreted during insect cell growth. Secretion of 4WCbac was found to be dependent on N-glycosylation at N313 but not at N85 and elimination of one or both of these sites appeared to have little influence on apyrase activity. In addition, both 4WCbac and 6RG:Hisbac from the cellular fraction were fully functional. These results were compared with similar work performed on the animal ecto-apyrases which have different specific N-glycosylation sites required for secretion and activity. The 4WCbac-sup, 7WCcoli and 6RG:Hisbac proteins all showed apyrase activity, that is they catalysed the hydrolysis of nucleotide tri- and/or di-phosphates to their corresponding nucleotide monophosphates, and released inorganic phosphate in a divalent cation-dependent manner. However, the proteins exhibited different activities, substrate specificities, pH profiles and influence of inhibitors: 4WCbac-sup had a preference for NDPs with a pH optimum ≥9.5; 7WCcoli had a modest preference for NTPs with a pH optimum at 8.5; 6RG:Hisbac was almost exclusively an NTPase with a pH optimum at 6.5. Contrary to predictions based on phylogeny the proteins all bound to sulphated disaccharides and their catalytic activities were influenced both positively and negatively by the binding of specific chitosans. The data indicates that all three soluble plant apyrases investigated here were LNPs, in contrast to predictions from the literature. In order to pinpoint the regions responsible for determining substrate specificity and chitosan binding, chimeras were made using the N- and C-terminal halves of 4WC and 6RG. This resulted in fully functional reciprocal chimeras. Comparison of the apyrase activity for parents and chimeras, substrate specificity, optimal pH, influence of inhibitors on activity and effects of chitosans indicated that the C-terminus was responsible for determining substrate specificity. However, the influence of specific chitosans on the chimeras appeared to be dependent on both the N- and C-terminal portions of the proteins. In addition, chimeras were found to bind to the same sulphated disaccharides as the parent proteins. Preliminary crystal screening experiments were performed with highly purified preparations of 7WCcoli and 6RG:Hisbac. Under specific conditions 7WCcoli was found to form cube-like crystalline arrangements while 6RG:Hisbac formed hexagonal-like crystalline structures. A potential model for carbohydrate binding by LNPs is proposed and the possible biological roles of plant LNPs are discussed.