Amyotrophic Lateral Sclerosis (ALS) is an adult-onset progressive neurodegenerative disease that causes degeneration in both upper and lower motor neurons. ALS progresses relentlessly after the onset of the disease, with most patients die within 3-5 years of diagnosis, largely due to respiratory failure. Since SOD1 became the first gene whose mutations were associated with ALS in 1993, more than 17 ALS causative genes have been identified. Among them, TAR DNA-binding protein (TARDBP) lies in the central of ALS pathology mechanism study, because TDP43 proteinopathy is observed not only in familial ALS cases carrying TARDBP mutations, but also in most of the sporadic ALS cases, which account for 90% of the whole ALS population. Several TDP43 overexpression mouse models have been successfully generated to study the gain-of-toxicity mechanism of TDP43 in ALS development, while the investigation of loss-of-function mechanism which could also contribute to ALS still awaits a proper mouse model. The major difficulty in generating TARDBP knock out mouse model lies in the fact that TARDBP is a development essential gene and complete depletion of TDP43 function causes embryonic lethality. In chapter I, I reviewed the recent advances in ALS study. Emphasis was given to ALS mouse models, especially TARDBP ALS mouse model. In Chapter II, I made a Tet-responsive construct that contains mCherry, a fluorescent protein, as an indicator for the expression of the artificial miRNA (amiTDP) residing in the 3’UTR of mCherry and targeting TARDBP. The construct was tested in NSC34 cells and TRE-mCherry-amiTDP43 transgenic mouse was generated with this construct. Crossing TRE-mCherry-amiTDP43 mouse with mPrp-tTA mouse, mCherry expression was successfully induced in mouse forebrain and cerebellum, but not in other tissues including spinal cord. By quantitative real-time PCR, amiTDP43 expression was confirmed to be coupled with mCherry expression. Fluorescent immunostaining revealed that mCherry was expressed in neurons, but not in astrocytes or microglia cells, and that in mCherry positive cells, TDP43 was significantly knocked down. Results from Nissl staining and GFAP immunostaining suggested that decrease of TDP43 in forebrain neuron only was not sufficient to cause neurodegeneration and neuron loss. In chapter III, I investigated the function of Armadillo Containing Protein 4 (ARMC4), which was originally considered ALS causative gene. Our study of the function of CG5155, the possible homolog of ARMC4 in Drosophila, indicated that CG5155 is a male fertility gene that is involved in spermatogenesis. Therefore, we have named this gene Gudu. The transcript of Gudu is highly enriched in adult testes. Knockdown of Gudu by a ubiquitous driver leads to defects in the formation of the individualization complex that is required for spermatid maturation, thereby impairing spermatogenesis. Furthermore, testis-specific knockdown of Gudu by crossing the RNAi lines with Bam-Gal4 driver is sufficient to cause the infertility and defective spermatogenesis. Since Gudu is highly homologous to vertebrate ARMC4, also an Armadillo-repeat-containing protein enriched in testes, our results suggest that Gudu and ARMC4 is a subfamily of Armadillo-repeat containing proteins with an evolutionarily conserved function in spermatogenesis.

This study used photovoice methodology to explore how people with early-stage dementia use their perceived strength and resources to cope with the illness. Purposive sampling was used to recruit participants. Thirteen women and seven men age 57 to 90 (mean 73.4) with MMSE scores ranging from 20 to 28 (mean 25.6) participated in the project. Participants were provided with a disposable camera and invited to take photographs relevant to their well-being. The photographs provided the means for participants to take the lead in dialogue sessions about their coping response. Grounded theory analysis revealed that initially, early-stage dementia precipitated a disruption in well-being. For the participants in this study, learning to live with the illness was an ongoing, continually shifting process of adjustment, where the participants moved from a familiar sense of well-being to an alternative sense of well-being. In this process the participants used a variety of strengths and resources ranging from those that supported them in reconstructing identity, regaining control, and rebuilding relationships to those that assisted them in reestablishing a healthy lifestyle and restoring meaning and purpose. Striving toward well-being while acknowledging the inconceivable notion of losing one's cognitive abilities was the central strength that the participants brought to this process. It captured the paradox the participants faced in living with early-stage dementia, which was to relegate the illness to the background of their lives while managing its symptoms in the foreground.

Topical exposure to isopropyl alcohol has been reported in the literature to be toxic if sufficient isopropyl alcohol is absorbed (1-5). A clinical case is reported where a 48-y-old female presented with multiple unexplained cardiac and neurological deficits. The woman had developed the deficits over a 6-mo period in which she had been soaking towels with isopropyl alcohol and applying then to her skin overnight to ease arm pain she was experiencing. Cessation of the isopropyl alcohol exposure resolved her deficits within 3 d. A controlled repeat dermal exposure to isopropyl alcohol under clinical observation reproduced the deficits noted with corresponding serum and urine concentrations of isopropyl alcohol and acetone. Cessation of topical isopropyl alcohol exposure lead to subsequent resolution of all toxicities.

Peripheral neuropathies are a set of common diseases that affect the peripheral nervous system, causing damage to vital connections between various parts of the body and the brain and spinal cord. Different clinical conditions are known to selectively impact various size nerve fibers, which often makes it difficult to diagnose which peripheral neuropathy a patient might have. The nerve conduction velocity diagnostic test provides clinically useful information in the diagnosis of some peripheral neuropathies. This method is advantageous because it tends to be minimally invasive yet it provides valuable diagnostic information. However, this test does not determine characteristics of peripheral nerve fiber size distributions, and therefore does not show any detailed information regarding the nerve fibers within the nerve trunk. Being able to determine which nerve fibers are contributing to the evoked potential within a nerve trunk could provide additional information to clinicians for the diagnosis of specific pathologies of the peripheral nervous system, such as chronic inflammatory demyelinating polyneuropathy or early diabetic peripheral neuropathy. In this study, three rat sciatic nerves are sectioned and stained with hematoxylin and eosin in order to measure the nerve fiber diameters within the nerve trunk. Stained samples are viewed using brightfield microscopy and images are analyzed using ImageJ. Histograms were created to show the frequency of various nerve fiber diameters. The nerve fiber diameters measured during this research are consistent with the range of previously published diameter values and will be used to support continuing research for a novel method to characterize peripheral nerve fiber size distributions using group delay.

<p>Systemic fungal infections brought about by <em>Cryptococcus</em> species are associated with some of the highest mortality rates of any infectious disease. Alarmingly these pathogens have overtaken tuberculosis as the second greatest killer among Sub-Saharan AIDS patients and are an emerging disease among immunocompetent populations on the Pacific Coast of North America. This clinical threat has been exacerbated by our inability to discover novel compounds that specifically target fungal cellular architecture at the genus level. To confront this challenge, we have made a concerted effort to biologically prospect the vast chemical potential of Actinomycete bacteria isolated from diverse and underexplored niches around the world. A novel phenotypic screen was developed whereby bacterial small molecule producers were co-cultured on agar plates in an intimate setting with evolutionary distant fungal pathogens <em>Candida albicans</em> and <em>Cryptococcus neoformans</em>. Diffusible small molecules released by the organisms created a signaling environment that stimulated profound phenotypic changes both in the Actinomycetes and the pathogens. We were able to discern a unique relationship whereby the growth of <em>C. neoformans</em> was specifically inhibited by Nigerian soil Actinomycete isolate curated as WAC 2288. Further bioactivity guided purification and chemical analysis lead to the identification of ibomycin, a previously undescribed 34 membered macrolactone decorated with seven sugar moieties. A draft genome of WAC 2288 revealed a 140kb gene cluster containing 12 type I PKS modules and downstream capacity to generate rare sugars are responsible for ibomycin biosynthesis. Purification of ibomycin analogs has revealed that the terminal vancosamine on the molecule is dispensable for bioactivity, establishing a chemical antecedent for target identification through affinity chromatography. Throughout these studies the unprecedented anticryptococcal activity of ibomycin is consistently recapitulated. Future work on the molecule may validate ibomycin as an effective antifungal therapy.</p> / Master of Science (MSc)

<p>Lectin-functionalized, Poly [oligo(ethylene glycol) methyl ether methacrylate] (<em>POEGMA</em>) loaded with 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA) and poly(ethylene glycol)–block-poly(D,L-lactic-co-glycolic acid) (PEG-PLGA) nanoparticles loaded with haloperidol were prepared with narrow size distributions and sizes < 135 nm. The microgels and nanoparticles exhibited high <em>Solanum tuberosum </em>lectin (STL) conjugation efficiencies, encapsulation efficiencies, and drug loading capacities. The <em>in vitro</em> release of PAOPA and haloperidol was slow in physiological conditions over 96 hours, demonstrating minimal drug leakage and the potential for efficient drug transport to the targeted brain tissue. POAPA, POEGMA and the STL-functionalized POEGMA microgels were found to be non-toxic in both cell lines, indicating that they would not be toxic when administered intranasally or when they reach the brain. The nasal epithelial cell uptake of rhodamine-labelled microgels was higher in cells when the STL-functionalization was present. All haloperidol-loaded nanoparticle formulations were found to be highly effective at inducing catalepsy, while intranasal administration of STL-functionalized nanoparticles using the intranasal spray device increased the brain tissue haloperidol concentrations by 2-3.5 fold compared to STL-functionalized particles administered intranasally with a pipette. For the first time, brain tissue concentrations of rhodamine-labelled microgels confirmed that microgels are capable of passing the blood-brain barrier and that this uptake is size dependent. These formulations demonstrate promise in the reduction of the drug dose necessary to produce a therapeutic effect with antipsychotic drugs for the treatment of schizophrenia using a non-invasive route of administration.</p> / Master of Science (MSc)

<p>Увод: Процена минималних неуролошких дисфункција (МНД) пружа информације о неуролошком стању детета, помаже у идентификовању вулнерабилности детета ка развоју моторних слабости, тешкоћа у учењу или поремећаја понашања. Деформитети кичменог стуба код деце предшколског узраста су у сталном порасту. Циљ истраживања: Утврђивање преваленце МНД-а у узорку предшколске деце тестирањем по Touwen-у, евалуација постуралног статуса код деце предшколског узраста, као и утврђивање повезаности јављања минималних неуролошких дисфункција и постуралних поремећаја код деце предшколског узраста. Материјал и методе: Истраживање је урађено у предшколској установи &bdquo;Радосно детињство&ldquo; из Новог Сада. Укупан узорак је обухватио 120-оро деце која су подељена у две групе у односу на узраст, 60-оро деце старости од 6 до 7 година (група А) и 60-оро деце старости од 5 до 6 година (група Б). Методом случајног избора су изабрана три вртића из различитих делова града, а деца су насумично одабрана са приближно једнаком заступљеношћу пола. Тестирање је урађено са Touwen-овим тестом модификованим од стране Hadders-Algre. Резултати су груписани у три групе: одсуство, присуство једноставних (присуство једне или две области дисфункције) и присуство комплексних МНД (присуство најмање три области дисфункције). Код све деце урађена је евалуација постуралног статуса, као и антропометријска мерења. Резултати: Постоји статистички значајна разлика у учесталости јављања МНД-а у односу на узраст, одступања су чешће присутна у млађем узрасту. МНД се чешће јављају код дечака у односу на девојчице, али није показана статистички значајна разлика. Код 68,3% деце уочени су елементи лошег држања тела. Најчешћа одступања од нормалног постуралног положаја била су у виду спуштених стопала (45,8%), асиметрије висине рамена (35%), искривљења ахилових тетива ван (30%), повећања слабинске кривине (29,2%) и асиметрије троуглова стаса (20%). Утврђена је статистички значајна позитивна повезаност између јављања минималних неуролошких дисфункција и лошег држања тела код деце предшколског узраста. Закључак: Деца старијих (узраст од 5 до 6 година) група предшколског узраста знатно чешће испољавају знаке минималне неуролошке дисфункције у односу на децу најстаријих (узраст од 6 до 7 година) група. Више од половине деце предшколског узраста показује одступање од нормалног постуралног статуса. Постоји статистички значајна позитивна повезаност између јављања минималних неуролошких дисфункција и лошег држања тела код деце предшколског узраста.</p> / <p>Uvod: Procena minimalnih neuroloških disfunkcija (MND) pruža informacije o neurološkom stanju deteta, pomaže u identifikovanju vulnerabilnosti deteta ka razvoju motornih slabosti, teškoća u učenju ili poremećaja ponašanja. Deformiteti kičmenog stuba kod dece predškolskog uzrasta su u stalnom porastu. Cilj istraživanja: Utvrđivanje prevalence MND-a u uzorku predškolske dece testiranjem po Touwen-u, evaluacija posturalnog statusa kod dece predškolskog uzrasta, kao i utvrđivanje povezanosti javljanja minimalnih neuroloških disfunkcija i posturalnih poremećaja kod dece predškolskog uzrasta. Materijal i metode: Istraživanje je urađeno u predškolskoj ustanovi &bdquo;Radosno detinjstvo&ldquo; iz Novog Sada. Ukupan uzorak je obuhvatio 120-oro dece koja su podeljena u dve grupe u odnosu na uzrast, 60-oro dece starosti od 6 do 7 godina (grupa A) i 60-oro dece starosti od 5 do 6 godina (grupa B). Metodom slučajnog izbora su izabrana tri vrtića iz različitih delova grada, a deca su nasumično odabrana sa približno jednakom zastupljenošću pola. Testiranje je urađeno sa Touwen-ovim testom modifikovanim od strane Hadders-Algre. Rezultati su grupisani u tri grupe: odsustvo, prisustvo jednostavnih (prisustvo jedne ili dve oblasti disfunkcije) i prisustvo kompleksnih MND (prisustvo najmanje tri oblasti disfunkcije). Kod sve dece urađena je evaluacija posturalnog statusa, kao i antropometrijska merenja. Rezultati: Postoji statistički značajna razlika u učestalosti javljanja MND-a u odnosu na uzrast, odstupanja su češće prisutna u mlađem uzrastu. MND se češće javljaju kod dečaka u odnosu na devojčice, ali nije pokazana statistički značajna razlika. Kod 68,3% dece uočeni su elementi lošeg držanja tela. Najčešća odstupanja od normalnog posturalnog položaja bila su u vidu spuštenih stopala (45,8%), asimetrije visine ramena (35%), iskrivljenja ahilovih tetiva van (30%), povećanja slabinske krivine (29,2%) i asimetrije trouglova stasa (20%). Utvrđena je statistički značajna pozitivna povezanost između javljanja minimalnih neuroloških disfunkcija i lošeg držanja tela kod dece predškolskog uzrasta. Zaključak: Deca starijih (uzrast od 5 do 6 godina) grupa predškolskog uzrasta znatno češće ispoljavaju znake minimalne neurološke disfunkcije u odnosu na decu najstarijih (uzrast od 6 do 7 godina) grupa. Više od polovine dece predškolskog uzrasta pokazuje odstupanje od normalnog posturalnog statusa. Postoji statistički značajna pozitivna povezanost između javljanja minimalnih neuroloških disfunkcija i lošeg držanja tela kod dece predškolskog uzrasta.</p> / <p>Introduction: Assessment of minor neurological dysfunction (MND) provides information about a child&#39;s neurological condition, which helps to identify the vulnerability of the child to the development of motor impairment, difficulties in learning or behavioral disorders. Spinal deformities in preschool children are constantly increasing. Aim: Determining the prevalence of MND in a sample of preschool children using Touwen&rsquo;s test, the evaluation of postural status in preschool children, as well as determining the relation between the occurrence of minor neurological dysfunction and postural disorders in preschool children. Material and methods: The examination was carried out in the preschool institution &ldquo;Radosno detinjstvo&rdquo; in the city of Novi Sad. The total sample included 120 children who were divided into two groups according to their age, 60 children aged 6 to 7 years (group A) and 60 children aged 5 to 6 years (group B). The children were recruited at three randomly selected kindergartens, and the children were randomly selected with approximately equal gender representation. The testing was done with Touwen&#39;s test modified by Hadders-Algra. The results were classified into three groups: the absence of MND, the presence of simple MND (presence of one or two domains of dysfunction) and the presence of complex MND (presence of at least three domains of dysfunction). Evaluation of postural status and anthropometric measurements were carried out for all the children. Results: There is a statistically significant difference in the prevalence of MND in relation to age, variations were more likely at a younger age. MND is more frequent in boys than in girls, but this difference is not statistically significant. In 68.3% of the children there are elements of bad posture. The most common deviations from the normal postural position are flat feet (45.8%), shoulder height asymmetry (35%), distortion of Achilles tendons to the outside (30%), excessive curvature of lower back (29.2%) and asymmetry of stature triangles (20%). There is a statistically significant positive correlation between the occurrence of minor neurological dysfunction and poor posture in children of preschool age. Conclusion: Children in older preschool groups (5 to 6 years old) more often show signs of minor neurological dysfunction in comparison to the children in the oldest groups (6 to 7 years old). More than half of the preschool children show deviation from normal postural status. There is a statistically significant correlation between the occurrence of minor neurological dysfunction and poor posture in children of preschool age.</p>

Genomic instability, in the form of gene mutations, insertions/deletions, and gene amplifications, is one of the hallmarks in many types of cancers and other inheritable genetic disorders. Trinucleotide repeat (TNR) disorders, such as Huntington’s disease (HD) and Myotonic dystrophy (DM) can be inherited and repeats may be extended through subsequent generations. However, it is not clear how the CAG repeats expand through generations in HD. Two possible repeat expansion mechanisms include: 1) polymerase mediated repeat extension; 2) persistent TNR hairpin structure formation persisting in the genome resulting in expansion after subsequent cell division. Recent in vitro studies suggested that a family A translesion polymerase, polymerase θ (Polθ), was able to synthesize DNA larger than the template DNA. Clinical and in vivo studies showed either overexpression or knock down of Polθ caused poor survival in breast cancer patients and genomic instability. However, the role of Polθ in TNR expansion remains unelucidated. Therefore, we hypothesize that Polθ can directly cause TNR expansion during DNA synthesis. The investigation of the functional properties of Polθ during DNA replication and TNR synthesis will provide insight for the mechanism of TNR expansion through generations.

The endoplasmic reticulum (ER) is a multi-functional cellular compartment that functions in protein folding, lipid biosynthesis, and calcium homeostasis. Perturbations to ER function lead to the dysregulation of ER homeostasis, causing the accumulation of unfolded and misfolded proteins in the cell. This is a state of ER stress. ER stress elicits a cytoprotective, adaptive signaling cascade to mitigate stress, the Unfolded Protein Response (UPR). As long as the UPR can moderate stress, cells can produce the proper amount of proteins and maintain a state of homeostasis. If the UPR, however, is dysfunctional and fails to achieve this, cells will undergo apoptosis. Diabetes mellitus is a group of metabolic disorders characterized by persistent high blood glucose levels. The pathogenesis of this disease involves pancreatic β-cell dysfunction: an abnormality in the primary function of the β-cell, insulin production and secretion. Activation of the UPR is critical to pancreatic β-cell survival, where a disruption in ER stress signaling can lead to cell death and consequently diabetes. There are several models of ER stress leading to diabetes. Wolcott-Rallison syndrome, for example, occurs when there is a mutation in the gene encoding one of the master regulators of the UPR, PKR-like ER kinase (PERK). In this dissertation, we show that Wolfram Syndrome 1 (WFS1), an ER transmembrane protein, is a component of the UPR and is a downstream target of two of the master regulators of the UPR, Inositol Requiring 1 (IRE1) and PERK. WFS1 mutations lead to Wolfram syndrome, a non-autoimmune form of type 1 diabetes accompanied by optical atrophy and other neurological disorders. It has been shown that patients develop diabetes due to the selective loss of their pancreatic β-cells. Here we define the underlying molecular mechanism of β-cell loss in Wolfram syndrome, and link this cell loss to ER stress and a dysfunction in a component of the UPR, WFS1. We show that WFS1 expression is localized to the β-cell of the pancreas, it is upregulated during insulin secretion and ER stress, and its inactivation leads to chronic ER stress and apoptosis. This dissertation also reveals the previously unknown function of WFS1 in the UPR. Positive regulation of the UPR has been extensively studied, however, the precise mechanisms of negative regulation of this signaling pathway have not. Here we report that WFS1 regulates a key transcription factor of the UPR, activating transcription factor 6 (ATF6), through the ubiquitin-proteasome pathway. WFS1 expression decreases expression levels of ATF6 target genes and represses ATF6-mediated activation of the ER stress response (ERSE) promoter. WFS1 recruits and stabilizes an E3 ubiquitin ligase, HMG-CoA reductase degradation protein 1 (HRD1), on the ER membrane. The WFS1-HRD1 complex recruits ATF6 to the proteasome and enhances its ubiquitination and proteasome-mediated degradation, leading to suppression of the UPR under non-stress conditions. In response to ER stress, ATF6 is released from WFS1 and activates the UPR to mitigate ER stress. This body of work reveals a novel role for WFS1 in the UPR, and a novel mechanism for regulating ER stress signaling. These findings also indicate that hyperactivation of the UPR can lead to cellular dysfunction and death. This supports the notion that tight regulation of ER stress signaling is crucial to cell survival. This unanticipated role of WFS1 for a feedback loop of the UPR is relevant to diseases caused by chronic hyperactivation of ER stress signaling network such as pancreatic β-cell death in diabetes and neurodegeneration.

Mutations in the SOD1 gene are the best characterized genetic cause of amyotrophic lateral sclerosis (ALS) and account for ~20% of inherited cases and 1-3% of sporadic cases. The gene-editing tool Cas9 can silence mutant genes that cause disease, but effective delivery of CRISPR-Cas9 to the central nervous system (CNS) remains challenging. Here, I developed strategies using canonical Streptococcus pyogenes Cas9 to silence SOD1. In the first strategy, I demonstrate effectiveness of systemic delivery of guide RNA targeting SOD1 to the CNS in a transgenic mouse model expressing human mutant SOD1 and Cas9. Silencing was observed in both the brain and the spinal cord. In the second strategy, I demonstrate the effectiveness of delivering both guide RNA and Cas9 via two AAVs into the ventricles of the brain of SOD1G93A mice. Silencing was observed in the brain and in motor neurons within the spinal cord. For both strategies, treated mice had prolonged survival when compared to controls. Treated mice also had improvements in grip strength and rotarod function. For ICV treated mice, we detected a benefit of SOD1 silencing using net axonal transport assays, a novel method to detect motor neuron function in mice before onset of motor symptoms. These studies demonstrate that Cas9-mediated genome editing can mediate disease gene silencing in motor neurons and warrants further development for use as a therapeutic intervention for SOD1-linked ALS patients.