More than 40 neurological diseases are known to be caused by large expansions oftandem repeat sequences scattered throughout the human genome in introns, exons and untranslated regions. The GGGGCC (G4C2) repeat expansion located in the first intron of the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). In C9 FTD/ALS, expanded transcripts are known to aggregate and accumulate in the cell nucleus, sequestering RNA binding proteins. Other expanded RNA species are exported to the cytoplasm to undergo a non-canonical form of translation termed ‘repeat-associated non-AUG (RAN) translation’. RAN translation leads to the production of toxic polydipeptide repeat proteins in the absence of a canonical AUG start codon. This dissertation will highlight new mechanistic features of translation across the G4C2 repeat expansion, identify a potential therapeutic for C9 FTD/ALS using RNAi and develop a cellular system to explore the G4C2 repeat RNA lifecycle. First, we demonstrate that increasing G4C2 repeat expansion size results in suppression of translation from both canonical and non-canonical start codons, suggesting that large polydipeptide repeats are rarely fully translated. We further find that initiation does not occur from within the repeat expansion, relying on upstream sequence for initiation. However, some reading frames are prone to substantial frameshifting, such as poly-GA. We also show that a bias in ii codon usage efficiency contributes to previously observed variations in the levels of each polydipeptide. Our results support and extend previous studies by identifying two new mechanisms that bias production of poly-dipeptides toward poly-GA in C9 FTD/ALS. Further, we generated central mismatch-containing short hairpin RNAs (shRNAs) targeting the G4C2 repeat expansion to reduce aggregation or block translation of repeatcontaining transcripts. Iterative design was able to improve shRNA processing efficiency and cellular abundance, yet they were unable to reduce nuclear RNA foci in patient-derived cells. Despite this, we show preliminary data suggesting that these shRNAs are able to target cytoplasmic repeat-containing transcripts and resulting in a reduced translation of poly-GP. Finally, we optimized the previously published RNA-protein interaction detection (RaPID) technique, which uses proximity dependent labelling by a mutant biotin ligase and mass spectrometry for protein identification in living cells, to identify proteins interacting with the G4C2 repeat expansion. We embedded the box B RNA hairpin between G4C2 repeats and tested the ability for λN fused to a biotin ligase mutant, BASU, to specifically bind the box B hairpin in vitro. We show that 6 repeats each side of the hairpin combined with an extended hairpin stem promotes specific binding of the λN-BASU fusion protein and is likely to be successful in cells. C9 FTD/ALS is a currently incurable neurodegenerative disorder largely due to the limited understanding of disease mechanism. This dissertation demonstrates new mechanisms of translation across the G4C2 repeat expansion that results in toxic DPR production while also developing a nucleic acid therapeutic for long-term treatment of C9 FTD/ALS and further developing systems to explore RNA-mediated toxicity in cells.

Neurodegenerative disorders are among the most devastating human illnesses. They present a significant source of morbidity and mortality, and given an aging population, an impending public health crisis. Disease-modifying treatments remain sparse, with most current therapies focused on reducing symptom burden. The cellular stress response is intimately linked to energy management and has frequently been posited as playing a central role in neurodegeneration. Using two distinct neurodegenerative diseases as ‘case studies’, aberrant cellular stress and energy management are demonstrated as potential pathways contributing to neurodegeneration. First, the Huntington’s disease protein, huntingtin, is observed to rapidly localize to early endosomes, where it is associated with arrest in early-to-late and early-to-recycling endocytic trafficking. Given the energy-dependent nature of vesicular trafficking, this arrest is postulated to free substantial energy within the cell, which may subsequently be diverted to pathways that are critical for the initiation of longer-duration stress responses, such as the unfolded protein response. In the context of Huntington’s disease, impaired recovery from this stress response is observed, suggesting deficits in intracellular vesicular trafficking and energy regulation exist in disease states. In the second ‘case study’, a novel spinocerebellar ataxia variant is characterized, occurring as a result of point mutations within two genes: ATXN7 and TOP1MT, which encode ataxin-7 and the type I mitochondrial topoisomerase (top1mt), respectively. Ataxin-7 has previously been implicated in spinocerebellar ataxia type 7, which occurs as a result of a polyglutamine expansion in the first exon of the protein. Patient cells are noted to have substantially lower mitochondrial respiratory function in comparison to healthy controls and decreased levels of mitochondrial DNA, and ataxin-7 subcellular localization is observed to be abnormal. This suggests that there is important interplay between the mitochondria and proteins implicated in neurodegeneration and provides further support for aberrant cellular bioenergetics as a unifying pathway to neurodegeneration. In the concluding chapters, the nuclear localization signal of ataxin-7 is characterized, and there is analysis comparing conical ‘atraumatic’ lumbar puncture needles with bevel-tipped ‘conventional’ needles. Atraumatic needles are noted to be associated with significantly less patient complications and require fewer return visits to hospital. Moreover, atraumatic needles are demonstrated to have similar rates of success and failure when controlling for important variables like clinician specialty, dispelling common misconceptions surrounding their ease-of-use. As lumbar puncture is ubiquitous within the clinical neurosciences and is important for diagnosis, monitoring, and treatment of disease, as well as clinical trials, this work has far-reaching implications for patient care and future research. / Thesis / Doctor of Philosophy (PhD)

Throughout the past decade American pine forests have experienced an epidemic of Mountain Pine Beetle (MPB) induced tree mortality. This thesis estimates the losses to home values caused by deteriorating forest quality in the Front Range Counties of Larimer and Boulder Colorado. We employ a repeat sales model that allows for region specific price indices, and non-linear age-related depreciation in home values. We use the time-invariant existence of pine forest near a home to overcome shortcomings in the measurement of MPB damage. We infer from temporal changes in the marginal "effect of pine trees near a home the approximate MPB "effect . We label this strategy the translating commodity approach. Using this strategy we are able to show that diminished forest quality causes forests to become a dis-amenity that negatively affects nearby home values. The total loss in 2011 home values due to their proximity to dying forest is estimated to be $137 million for all the homes in our sample. Such substantial losses may justify a forest management policy shift in order to better mitigate the risk of future MPB outbreaks. / Master of Science

Many applications in biomedicine, research, and industry require recognition agents with specificity and selectivity for their target. Protein engineering enables the design of scaffolds that can bind targets of interest while increasing their stability, and expanding the scope of applications in which these scaffolds will be useful. Repeat proteins are instrumental in a wide variety of biological processes, including the recognition of pathogen-associated molecular patterns by the immune system. A number of successes using alternative immune system repeat protein scaffolds have expanded the scope of recognition agents available for targeting glycans and glycoproteins in particular. We have analyzed the innate immune genes of a freshwater polyp and found that they contained particularly long contiguous domains with high sequence similarity between repeats in these domains. We undertook statistical design to create a binding protein based on the H. magnipapillata innate immune TPR proteins. My second research project focused on creating a protein to bind cellulose, as it is the most abundant and inexpensive source of biomass and therefore is widely considered a possible source for liquid fuel. However, processing costs have kept lignocellulosic fuels from competing commercially with starch-based biofuels. In recent years a strategy to protect processing enzymes with synergistic proteins emerged to reduce the amount of enzyme necessary for lignocellulosic biofuel production. Simultaneously, protein engineering approaches have been developed to optimize proteins for function and stability enabling the use of proteins under non-native conditions and the unique conditions required for any necessary application. We designed a consensus protein based on the carbohydrate-binding protein domain CBM1 that will bind to cellulosic materials. The resulting designed protein is a stable monomeric protein that binds to both microcrystalline cellulose and amorphous regenerated cellulose thin films. By studying small changes to the binding site, we can better understand how these proteins bind to different cellulose-based materials in nature and how to apply their use to industrial applications such as enhancing the saccharification of lignocellulosic feedstock for biofuel production. Biomaterials made from natural human hair keratin have mechanical and biochemical properties that make them ideal scaffolds for tissue engineering and wound healing. However, the extraction process leads to protein degradation and brings with it byproducts from hair, which can cause unfavorable immune responses. Recombinant keratin biomaterials are free from these disadvantages, while heterologous expression of these proteins allows us to manipulate the primary sequence. We endeavored to add an RGD sequence to facilitate cell adhesion to the recombinant keratin proteins, to demonstrate an example of useful sequence modification. / Doctor of Philosophy / Many applications in medicine and research require molecular sensors that bind their target tightly and selectively, even in complex mixtures. Mammalian antibodies are the best-studied examples of these sensors, but problems with the stability, expense, and selectivity of these antibodies have led to the development of alternatives. In the search for better sensors, repeat proteins have emerged as one promising class, as repeat proteins are relatively simple to design while being able to bind specifically and selectively to their targets. However, a drawback of commonly used designed repeat proteins is that their targets are typically restricted to proteins, while many targets of biomedical interest are sugars, such as those that are responsible for blood types. Repeat proteins from the immune system, on the other hand, bind targets of many different types. We looked at the unusual immune system of a freshwater polyp as inspiration to design a new repeat protein to recognize nonprotein targets. My second research project focused on binding cellulose, as it is the most abundant and inexpensive source of biological matter and therefore is widely considered a possible source for liquid fuel. However, processing costs have kept cellulose-based fuels from competing commercially with biofuel made from corn and other starchy plants. One strategy to lower costs relies on using helper proteins to reduce the amount of enzyme needed to break down the cellulose, as enzymes are the most expensive part of processing. We designed such a protein for this function to be more stable than natural proteins currently used. The resulting designed protein binds to multiple cellulose structures. Designing a protein from scratch also allows us to study small changes to the binding site, allowing us to better understand how these proteins bind to different cellulose-based materials in nature and how to apply their use to industrial applications. Biomaterials made from natural human hair keratin have mechanical and biochemical properties that make them ideal for tissue engineering and wound healing applications. However, the process by which these proteins are extracted from hair leads to some protein degradation and brings with it byproducts from hair, which can cause unfavorable immune responses. Making these proteins synthetically allows us to have pure starting material, and lets us add new features to the proteins, which translates into materials better tailored for their applications. We discuss here one example, in which we added a cell-binding motif to a keratin protein sequence.

Yes / Purpose: The chief objective of the study is to understand that how different demographic variables and repeated availing of service from the same doctor or same hospital shapes the overall perception of healthcare service quality and satisfaction among inpatients admitted in private hospitals in an emerging economy. Methodology: A self-administered, cross-sectional survey of inpatients using a questionnaire translated into Hindi and Gujarati. The data was collected from 702 inpatient from 18 private clinics located in three selected cities from Western India. Findings The results indicate that experience with hospital administration, doctors, nursing staff, physical environment, hospital pharmacy and physical environment is significant predictor of inpatient satisfaction. Physical environment was found to be significantly associated with satisfaction only among female inpatient. It was also found that repeat availing of services either from the same hospital or doctor does not increases patient satisfaction. The feasibility, reliability and validity of the instrument that measures major technical and non-technical dimensions of quality of healthcare services were established in the context of a developing country. Originality/Value: The study makes important contribution by empirically investigating the inpatient assessment of healthcare service quality based upon their demographic information and repeated availing of services to understand how repeat visit shapes the service quality perception.

• Argonaute proteins are important effectors in RNA silencing pathways, but they must interact with other machinery to trigger silencing. Ago hooks have emerged as a conserved motif responsible for interaction with Argonaute proteins, but little is know about the sequence surrounding Ago hooks that must restrict or enable interaction with specific Argonautes. • Here we investigated the evolutionary dynamics of an Argonaute-binding platform in NRPE1, the largest subunit of RNA Polymerase V. We compared NRPE1 sequences from more than 50 species, including dense sampling of two plant lineages. • This study demonstrates that the Argonaute-binding platform of NRPE1 retains Ago-hooks, intrinsic disorder, and repetitive character while being highly labile at the sequence level. We reveal that loss of sequence conservation is due to relaxed selection and frequent expansions and contractions of tandem repeat arrays. These factors allow a complete restructuring of the Ago-binding platform over 50-60 million years. This evolutionary pattern is also detected in a second Ago-binding platform, suggesting it is a general mechanism. • The presence of labile repeat arrays in all analyzed NRPE1 Ago-binding platforms indicates that selection maintains repetitive character, potentially to retain the ability to rapidly restructure the Ago-binding platform.

La RNase P est impliquée dans la maturation des ARNt en libérant l’extrémité 5’ leader des précurseurs d’ARNt. Jusqu’à récemment, il était admis que cette enzyme soit universellement conservée en tant que complexe ribonucléoprotéique. La rupture avec le modèle établi est venue avec l’identification d’une RNase P uniquement protéique dans les mitochondries humaines et chez les plantes. Ces protéines, nommées PRORP (PROteinaceous RNase P), présentent trois paralogues chez Arabidopsis, qui sont localisés soit dans les organelles (PRORP1), soit dans le noyau (PRORP2 et 3). Des tests d’activité in vitro montrent que les protéines PRORP possèdent seules une activité RNase P. L’étude de lignées ADN-T indique que la fonction des protéines PRORP est essentielle et la fonction de PRORP2 et 3 est redondante. L’analyse des lignées de dérégulation montre que les protéines PRORP possèdent une diversité de substrat et que la RNase MRP, une autre ribonucléoprotéine, n’est pas impliquée dans la maturation des ARNt. Ainsi les protéines PRORP seraient bien les seules enzymes impliquées dans la maturation de l’extrémité 5’ des ARNt chez Arabidopsis. / RNase P is involved in the maturation of tRNA precursors by cleaving their 5’ leader sequences. Until recently this enzyme was considered to be universally occurring as a ribonucleoprotein complex. The breakthrough from the existing model came with the identification of protein-only RNase P in human mitochondria as well as in plants. These proteins that we called PRORP (PROteinaceous RNase P) have three paralogs in Arabidopsis, which are localised in organelles (PRORP1) and nuclei (PRORP2 and 3). We have shown that PRORP proteins have RNase P activity in vitro as single proteins. In vivo the functions of PRORP proteins are essential and the function of PROPR2 and 3 are redundant. PRORP down-regulation mutants, show that PRORP proteins have a variety of other substrates and RNase MRP, another ribonucleoprotein, is not involved in the tRNA maturation. Results show that PRORP proteins would be the only enzymes responsible for RNase P activity in Arabidopsis.

Trinucleotide repeat (TNR) instability including repeat expansions and repeat deletions is the cause of more than 40 inherited incurable neurodegenerative diseases and cancer. TNR instability is associated with DNA damage and base excision repair (BER). In this dissertation research, we explored the mechanisms of BER-mediated TNR instability via biochemical analysis of the BER protein activities, DNA structures, protein-protein interaction, and protein-DNA interaction by reconstructing BER in vitro using synthesized oligonucleotide TNR substrates and purified human proteins. First, we evaluated a germline DNA polymerase β (pol β) polymorphic variant, pol βR137Q, in leading TNR instability-mediated cancers or neurodegenerative diseases. We find that the pol βR137Q has slightly weaker DNA synthesis activity compared to that of wild-type (WT) pol β. Because of the similar abilities between pol βR137Q and WT pol β in bypassing a template loop structure, both pol βR137Q and WT pol β induces similar amount of repeat deletion. We conclude that the slightly weaker DNA synthesis activity of pol βR137Q does not alter the TNR instability compared to that of WT pol β, suggesting that the pol βR137Q carriers do not have an altered risk in developing TNR instability-mediated human diseases. We then investigated the role of DNA synthesis activities of DNA polymerases in modulating TNR instability. We find that pol βY265C and pol ν with very weak DNA synthesis activities predominantly promote TNR deletions. We identify that the sequences of TNRs may also affect DNA synthesis and alter the outcomes of TNR instability. By inhibiting the DNA synthesis activity of pol β using a pol β inhibitor, we find that the outcome of TNR instability is shifted toward repeat deletions. The results provide the direct evidence that DNA synthesis activity of DNA polymerases can be utilized as a potential therapeutic target for treating TNR expansion diseases. Finally, we explored the role of post-translational modification (PTM) of proliferating cell nuclear antigen (PCNA) on TNR instability. We find that ubiquitinated PCNA (ub-PCNA) stimulates Fanconi associated nuclease 1 (FAN1) 5’-3’ exonucleolytic activities directly on hairpin structures, coordinating flap endonuclease 1 (FEN1) in removing difficult secondary structures, thereby suppressing TNR expansions. The results suggest a role of mono-ubiquitination of PCNA in maintaining TNR stability by regulating nucleases switching. Our results suggest enzymatic activities of DNA polymerases and nucleases and the regulation of the activities by PTM play important roles in BER-mediated TNR instability. This research provides the molecular basis for future development of new therapeutic strategies for prevention and treatment of TNR-mediated neurodegenerative diseases.

Nesta tese são apresentados um novo modelo estatístico para cálculo de probabilidade de paternidade e sua implementação em software. O modelo proposto utiliza o genótipo como informação básica, em contraste com outros modelos que usam alelos. Por esta diferença, o modelo proposto resulta mais abrangente, mas que, sob certas restrições, reproduz os resultados dos modelos que usam alelos. Este modelo foi implementado em um software que recebe descrições da genealogia e dos marcadores em uma linguagem dedicada a isso e constrói uma rede bayesiana para cada marcador. O usuário pode definir livremente a genealogia e os marcadores. O cálculo da probabilidade de paternidade é feito, sobre as redes construídas, por um software para inferência em redes bayesianas e a probabilidade de paternidade combinada considerando todos os marcadores é calculada, resultando em um \"índice de paternidade. / This thesis presents a novel statistical model for calculation of the probability of paternity and its implementation as a software. The proposed model uses genotype as basic information. Other models use alleles as basic information. As a result the proposed model is broader, in the sense that, under certain constraints the results from the other models are reproduced. The software implementation receives pedigree and markers data, in a specifically designed language, as input and builds one bayesian network for each marker. The user can freely define any pedigree and any marker. Paternity probabilities for each locus are calculated, from the built networks, by a software for inference on Bayesian Networks and these probabilities are combined into a single \"paternity index\".

Afin de caractériser les mécanismes moléculaires sous-jacents au processus d’adhésion des bactéries commensales, nous avons utilisé Streptococcus salivarius comme modèle. Streptococcus salivarius est une bactérie pionière dans la colonisation des surfaces orales chez le nouveau né, et devient par la suite un composant majoritaire du microbiote oral de l'adulte avec un rôle écologique majeur. Nous avons développé une méthode pour identifier, par des tests de criblage phénotypique, les gènes impliqués dans l’adhésion de S. salivarius aux surfaces bactériennes ou de l’hôte. Notre approche a permis d’identifier un ensemble de gènes codant pour des protéines de surfaces, des glycosyltransférases, des transporteurs qui sont impliqués dans les phénomènes d’auto-agréation et / ou de co-agrégation avec d’autres espèces et / ou l’adhésion aux protéines de l’hôte.En particulier, nous avons montré que le système SecA2Y2, qui comprend des gènes codant pour des protéines dédiées à la glycosylation et l'export de protéines de surface riche en sérine (SRRPs), participe aux processus d’agrégation, de formation de biofilms, à l'adhésion in vitro aux protéines de l’hôte et in vivo à la colonisation du tractus digestif de souris. Alors que toutes les bactéries contenant un système similaire possèdent un substrat unique au système, une SRRP, le locus génétique secA2Y2 comprend trois SRRPs qui présentent des rôles complémentaires dans les phénotypes précédement cités. SrpB est spécifiquement impliquée dans la liaison aux cellules epitheliales, tandis que SrpC participe à l’adhésion aux protéines de la matrice extracellulaire et le mucus. De manière atypique, nous avons démontré que le processus de maturation des SRRPs est supporté par glycosyltransférases extra-cluster. Cette étude est le premier rapport indiquant la présence dans une bactérie de trois SRRPs, qui présentent des rôles complémentaires dans l'interaction bactéries-hôte. Bien que le système SecA2Y2 soit principalement associé à la virulence des bactéries pathogènes, il semble être clairement impliqué dans les caractères de commensalité de S. salivarius, tels que la colonisation de ses niches écologiques orales et intestinales. Ce travail offre de nouvelles perspectives sur les mécanismes de colonisation des bactéries commensales. / To characterize molecular mechanisms underlying adhesion of commensal bacteria, we used Streptococcus salivarius (SSAL) as a model. SSAL is among the most important pioneer colonizers of neonatal oral mucosal surfaces, and later becomes a predominant component of the human adult oral microbiota with pre-eminent ecological role. We developed a method to identified, through phenotypic screening assays, genes involved in SSAL adhesion to host or bacterial surfaces. In particular, we showed that the SecA2Y2 system, which comprises genes devoted to glycosylation and export of surface Serine Rich Repeat Proteins (SRRPs), participates to bacterial aggregation, biofilm formation, in vitro adhesion and colonization of mice. While all bacteria containing a similar system possess only one SRRP, the SSAL secA2Y2 locus comprises three SRRPs with complementary role in line with the previous phenotypes. Interestingly, SrpB is specifically involved in the binding to epithelial cells, while SrpC to the extracellular matrix and mucus proteins. We showed that these interactions require glycosylation of both bacterial SRPs and host surfaces. Surprisingly, we demonstrated that this essential process is shared by glycosyltransferases located in other genomic regions. This work is the first report showing the presence in a bacterium of three SRPs, which display complementary roles in bacterial-host interaction. While the SecA2Y2 system is mostly associated to virulence in pathogenic bacteria, it appears to be involved in the expression of commensal traits in SSAL, such as its colonization and its resilience to oral and intestinal niches. This work may offer new insights into the mechanisms of niche establishment (host, microbial communities) of commensal bacteria.