Thesis (Ph. D.)--University of Chicago, Dept. of Chemistry, March 2003. / Includes bibliographical references. Also available on the Internet.

1. Transfection is a key technique in cell and molecular biology with many important biochemical applications. We selected a fiber fs laser at 1554 nm, an instrument widely used in optical communication research, as the excitation source. Our results demonstrated that the fs laser could perforate the cell membrane and the hole would close in sub-second interval after the laser exposure. We determined the safe exposure duration by detecting if there was any sign of mitochondrial depolarization at 1.5 hours after photoporation. Furthermore, we had successfully transfected HepG2 cells with a plasmid DNA containing the OFP gene, whose fluorescence could still be detected 24 hours after exposure. The transfection efficiency was as high as 77.3%. We also observed the proliferation of the transfected cells after 48 hours. / 2. Cell-cell fusion is a powerful tool for the analysis of gene expression, chromosomal mapping, monoclonal antibody production, and cancer immunotherapy. One of the challenges of in vitro cell fusion is to improve the fusion efficiency without adding extra chemicals while maintaining the cells alive and healthy. We show here that targeted human cancer cells could be selected by an optical tweezer and fused by a finely focused fs laser beam at 1554 nm with a high fusion eftlciency. The result confirmed that human cells could be fused exclusively by fs laser pulses, and this is the first time human cells are fused together all-optically. Mixing of cytoplasm in the fused cells was subsequently observed, and cells from different cell lines were also fused. Based on these, we firstly developed the method of optical cell-cell fusion. / 3. Failure in the induction of apoptosis or programmed cell death is one of the major contributions to the development of cancer and autoimmune diseases. Here we used a fs laser as a novel method to provide a direct apoptosis trigger to observe dynamic changes at subcellular level during apoptosis. First, we examined the effect of fs laser irradiation on the creation of reactive oxygen species (ROS) in exposed cells, which could trigger programmed cell death. By controlling the mitochondria electron transport chain (ETC), we investigated the mechanism of ROS generation by the fs pulses, including thermal effect and direct free electron liberation. Second, we induced apoptosis to targeted cells by the fs laser and found that the nuclear envelope (NE) formed tubular or tunnel-like structures (nuclear tubules - NT) inside the nucleus. The average number of NTs in each cell with laser treatment was significantly larger than in the control. Besides, the development of a NT was observed since its inception and it eventually merged with another one to form a larger NT. Meanwhile, mitochondria and tubulin were found inside the NT, and the NT formation always occurred after an upsurge of cellular Ca2+ concentration. More DNA fragmentation were also found in the region around the NTs. Based on this, we propose that NTs are developed during apoptosis and mitochondria migrate into the nucleus through the NTs to release death signals to trigger DNA fragmentation. Third, we used the fs laser to induce Ca2+ in cells in the form of a slow release, and firstly discovered that most Ca2+ was stored in the cytoplasm, and could diffuse into the nucleus after the optical trigger. Using fast confocal scanning, we obtained the path way of Ca2+ diffusion after the trigger in different cases. Our findings thus provide a new method of regulating the rate of apoptosis. / Biophotonics is an exciting and fast-expanding frontier which involves a fusion of advanced photonics and biology. It has not only developed many novel methodologies for biomedical research, but also achieved significant results as an independent field. Aided with femtosecond (fs) laser technologies, important progresses have been made on manipulating, imaging, and engineering of biological samples from single molecules to tissues in the last 10 years. The laser beam of ultra-short pulses at near-infrared band enjoys a lot of advantages: high nonlinear efficiency, low absorption by biological samples, high spatial and temporal resolution with tight confinement, low photo-toxicity, non-invasive, and ease of control. In this thesis, we report new findings from cell manipulation by fs laser, including transfection, cell-cell fusion, and induction of apoptosis in cells, which are detailed as follows: / He, Hao. / Adviser: Kam Tai Chan. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

In this thesis, experiments into artificially guiding neuronal growth cones using tightly focused laser beams were performed and evaluated. The experiments are performed by focusing a laser beam to the leading edge of a developing growth cone and attempting to change the direction of growth cone. These experiments were carried out using Gaussian, line and asymmetric line beam profiles. There was no noticeable change in the success rate with different beam profiles. Following this, I assisted my colleague Dr Michael Mazilu in the construction of a mathematical model of filopedia in an optical field in order to help explain the mechanism for optically guided neuronal growth which suggests that optical trapping forces on filopedia are responsible. Next, I set about implementing a system to automate the process of laser guided neuron growth by employing a spatial light modulator and a custom-built computer program. This allowed the computer to track a developing growth cone and automatically adjust the position of the laser beam as the growth cone developed. This program was successfully employed to artificially grow neuronal growth cones towards a user-inputted target point. The use of the spatial light modulator to beam shape was also demonstrated with the use of a Bessel beam being used to guide neurons for the first time. I also used a transgenic cell line of neurons to show for the first time that HSP70 is not involved in this phenomenon. This was accomplished by transfecting NG108’s with a plasmid containing HSP70 promoter tagged GFP. Under enough thermal or mechanical stress, the cells would express HSP70 which would produce a detectable GFP signal. No GFP was detected in cells after being exposed to laser irradiation of a power higher than would normally be used to guide neurons. Combined, these experiments show that the beam profile of the operating laser does not significantly affect the success of artificial growth and that the optical force on filopedia near the laser beam is likely to be the mechanism for this phenomenon. A possible heating effect of the laser has also been shown to not be strong enough to elicit a heat shock stress response from the cell. The demonstration of an automatic system which incorporates beam shaping has also been shown and such a system shows the potential to advance the investigation of artificial neuron growth using lasers.

Hydrogen is universally known as the most efficient renewable energy source capable of meeting global energy demands. Chlamydomonas reinhardtii has the ability to produce biohydrogen during the metabolic engineering of the photosynthetic pathways. The aim of this study was to 1) use leachate as a feedstock to enhance microalgal biomass and subsequent hydrogen production, 2) use Pulse Amplitude Modulated (PAM) Fluorometry to elucidate the role of photosystem one during hydrogen production, 3) use Nicotinamide Adenine Dinucleotide Phosphate (NADPH) fluorescence as an indicator of hydrogen production and 4) design a modular pilot scale biohydrogen bioprocessing system implementing experimental findings into a conceptual model. This resulted in a cost effective source of renewable hydrogen produced from waste. The use of 16% landfill leachate was found to increase biomass production by 26% as compared to using Tris- Acetate Phosphate (TAP) media alone. Hydrogen induction resulted in an increased gas synthesis of 37% as well as an increased production period of 8 days compared to the normal 5 days. Landfill leachate further reduced the costs as it acted as a free nutrient source with the added ecological advantage of leachate treatment. Hydrogen production was induced by sulphur depletion and physiological parameters were measured using PAM Fluorometry. Photosystem I was found to be dominant during hydrogen production while photosystem II was down-regulated due to the sulphur depletion and damaged D1 proteins. NADPH fluorescence was significantly correlated to hydrogen yields allowing for NADPH to be utilised as a molecular indicator for hydrogen synthesis. The overall functionality of this bioprocessing system relies on the optimum physiological functioning of cells. The above findings were implemented into a pilot scale design, maximising the physiological performance during hydrogen production. This study has contributed knowledge regarding the production of hydrogen gas from leachate, the physiological changes of photosystem I during hydrogen production and the use of NADPH fluorescence as an indicator. The fundamental theories of bioprocessing incorporate a firm understanding of cellular and biochemical processes. The use of molecular indicators determined from physiological studies can be used at pilot scale to improve overall efficiency of hydrogen production. / Ph.D. University of KwaZulu-Natal, Durban 2014.

Thesis (Ph.D.) - University of St Andrews, March 2007.

Thesis (Ph.D.) - University of St Andrews, October 2007.

Oligotrophic (sub-)tropical oceans receive intense incident ultraviolet radiation (UV, 280–400 nm) and their water columns are highly transparent due to their nutrient-deficient state. This combination suggests a high potential for adverse effects on organisms, yet only few reports describe the UV exposures received in these waters and the associated impacts on marine biota. Here, we aimed to investigate the UV bio-optics of various open ocean locations and, using the Red Sea as a representative oligotrophic environment, we investigated the pattern of UV attenuation over a wide latitudinal range, quantified UV exposures in the water column, and determined impacts of UVB (280–320 nm) on indigenous phytoplankton and scleractinian corals. Globally, the lowest average downwelling diffuse attenuation coefficients (Kd) in the UV spectrum were recorded in the ultra-oligotrophic Indian Ocean Subtropical Gyre (Kd(313nm): 0.110 m-1) and South Pacific Gyre (Kd(313nm): 0.098 m-1), while aCDOM(λ) was ~1–2 orders of magnitude higher than ap(λ), In the Red Sea, UV attenuation mirrored the prevailing latitudinal gradient in nutrients, with the lowest and highest Kd(313) of 0.130 m-1 and 0.357 m-1 measured in the far north and in the south of the basin, respectively. Central Red Sea waters were most transparent to UV in late summer, i.e., a few weeks after incident irradiances and SSTs reach their annual maximum. Although, the projected increase of SST due to climate change means that extreme UV exposure and temperatures could coincide in the near future. This finding is of particular relevance since we found that Red Sea diatom species such as C. closterium are highly sensitive to UVB-induced photoinhibition and cell decay (LRD50: 11.4 kJ). Water temperature also governed the UVB sensitivity of Synechococcus sp., although this group exhibited a high resistance overall (LRD50: 57 kJ to non-detectable). For corals, we found that UVB-removal generally had little impact on the oxidative stress levels and photophysiology of S. pistillata and P. verrucosa from shallow waters, but considerably accelerated the acclimation of upward transplanted corals, which highlights that UVB is a crucial stressor that governs the photoacclimation capacity of Red Sea corals.

Earth’s organisms are exposed to day-night cycles. These periodic changes in environmental factors, such as temperature and light exposure, trigger regulatory processes that coordinate physiological adaptations in organisms. Circadian organisms, i.e., most eukaryotes and some phototrophic bacteria, undergo autonomous 24-hour biological rhythms that are synchronized to day-night cycles via sensing light cues. However, the extent to which non-phototrophic bacteria tune their physiology to diurnal cycles and exhibit rhythmic behavior has been underexplored. For my thesis work, I investigated how the chemotrophic bacterium Pseudomonas aeruginosa responds to light and temperature signals. This metabolically versatile bacterium regulates its physiology through a vast array of environmental sensing mechanisms and has evolved multiple strategies to cope with redox imbalances. This thesis seeks to address how P. aeruginosa coordinates its metabolic and redox-balancing programs in response to light and temperature changes that occur in its environmental niche. In Chapter 1, I will present background information on relevant concepts such as biological rhythms and photosensory mechanisms and discuss how these principles are connected to physiological adaptations and metabolic plasticity in both phototrophic and non-phototrophic organisms, with a specific focus on chemotrophic bacteria. In Chapter 2, I will demonstrate that P. aeruginosa biofilm development is attenuated by light and that this process is regulated by the integration of light and redox signals. My work presented in Chapter 3 will provide evidence that the transcriptomic and metabolic landscape of P. aeruginosa is vastly reorganized in response to light/dark cycles. In the Chapter 4, I will explore how this reprogramming is manifested through activity by the respiratory machinery and I will demonstrate that P. aeruginosa undergoes intrinsic respiratory oscillations. As an opportunistic pathogen, P. aeruginosa will experience circadian-controlled changes during infection of a (circadian) host through host immune activity as well as exposure to cyclic environmental factors like light and temperature. I will discuss how environmental sensing is relevant for P. aeruginosa’s adaptation to its host-associated lifestyle. In conclusion, the research presented in this thesis establishes that P. aeruginosa exhibits an intricate physiological response to environmental signals, particularly light and temperature. This thesis contributes to a growing body of work that underscores how bacteria have evolved intricate mechanisms to integrate information about their environmental habitat, including host-associated conditions.

Yes / Photobiomodulation is reported to positively influence hair regrowth, wound healing, skin rejuvenation, and psoriasis. Despite rapid translation of this science to commercial therapeutic solutions, significant gaps in our understanding of the underlying processes remain. The aim of this review was to seek greater clarity and rationality specifically for the selection of optical parameters for studies on hair regrowth and wound healing. Our investigation of 90 reports published between 1985-2015 revealed major inconsistencies in optical parameters selected for clinical applications. Moreover, poorly understood photoreceptors expressed in skin such as cytochrome c oxidase, cryptochromes, opsins, may trigger different molecular mechanisms. All this could explain the plethora of reported physiological effects of light. To derive parameters for optimal clinical efficacy of photobiomodulation, we recommend a more rational approach, underpinning clinical studies with research of molecular targets and pathways using well-defined biological model systems enabling easy translation of optical parameters from in vitro to in vivo. Furthermore, special attention needs to be paid when conducting studies for hair regrowth, aiming for double-blind, placebo-controlled randomized clinical trials as the gold standard for quantifying hair growth. / European Marie-Curie Actions Programme, Grant agreement no.: 607886

O óxido nítrico (NO) é um mensageiro biológico que tem importância vital em muitos processos fisiológicos, tais como o controle cardiovascular, sinalização neural e defesa contra microrganismos e tumores. No entanto, a formação de outras espécies reativas, resultantes de reações químicas do NO com o ambiente biológico, impõe limites para o entendimento dos mecanismos celulares envolvidos em possíveis respostas biológicas. Devido ao potencial farmacológico e aos benefícios do NO, é de interesse o desenvolvimento de compostos que, quando estimulados, possam liberar esta molécula de forma controlada. Uma das possibilidades envolve complexos rutênionitrosilo termodinamicamente estáveis, mas que possam ser ativos sob estimulação. Este trabalho apresenta observações recentes de complexos rutênio-nitrosilo como agentes liberadores de NO e seus efeitos sobre linhagens de células B16-F10, L929 e Jukart. Para entender melhor o efeito NO como agente anticancerígeno o complexo [Ru(NO)(bdqi)(terpy)]Cl3 foi utilizado como agente doador de NO. O efeito biológico do composto, bem como o do seu produto após liberação de NO - [Ru(H2O)(bdqi)(terpy)]2+- foi avaliado pelos aspectos químicos e fotoquímicos. Os resultados mostraram que o NO, oriundo deste sistema, apresentou baixa citotoxicidade em células B16-F10. A atividade citotóxica foi maior quando o complexo foi encapsulado em nanopartículas lipídicas sólidas, diminuindo a viabilidade celular para cerca de 50 %. Além disso, sugere-se um efeito sinérgico da espécie aquo-rutênio, cuja viabilidade celular foi diminuída para 25% após 24 horas de incubação com este complexo. O efeito sinérgico do oxigênio singleto e NO foi também avaliado mediante a possibilidade de aplicação em terapia clínica. A produção de espécies radicalares de oxigênio tem sido utilizada para o tratamento do câncer, numa técnica conhecida como terapia fotodinâmica (TFD). O sucesso dessa terapia depende da concentração de oxigênio, e quando em hipóxia, geralmente culmina na formação reduzida de espécies reativas de oxigênio e consequente limitação clínica da TFD. Um dos objetivos deste trabalho é a avaliação do efeito sinérgico entre oxigênio singleto e NO. Para fins destes estudos, a espécie trinuclear [{Ru(NO)(bpy2)}2RuPc(pz)2](PF6)6 (I) foi sintetizada e proposta como fotogeradora de oxigênio singleto e NO. Ensaios fotobiológicos utilizando (I) a 0,4 ?M, em células B16-F10, resultaram na diminuição da viabilidade celular para 30% sob irradiação luminosa em 660 nm, ao final de 4 horas. Por outro lado, sem fotoestímulo e na mesma concentração do composto (I), a viabilidade celular foi de 90%. Estudos relacionados ao mecanismo de morte celular em consequência da ação do oxigênio singleto e NO gerados pelo composto (I) também foram discutidos neste trabalho. A possibilidade de aplicação de um sistema como o descrito em (I) no tratamento contra o câncer pode ser considerado interessante na terapia fotodinâmica / Nitric oxide (NO) is a biological messenger that has vital importance in many physiological processes, such as cardiovascular control, the neural signaling and defense against microorganisms and tumors. However, the formation of other reactive species, resulting from chemical reactions of NO with the biological environment, imposes limits on the understanding of the possible cellular mechanisms involved in biological responses. Due to potential pharmacological and benefits of NO, there is a need for development of compounds that can stabilize the NO until it\'s released. One possibility involves nitrosyl ruthenium complexes thermodynamically stable but actived under stimulation. In this work, the focus is on our recent investigations of nitrosyl ruthenium complexes as NO-delivery agents and their effects on B16-F10, L929 and Jukart cell lines. The high affinity of ruthenium for NO is a marked feature of its chemistry. To better understand the NO effect as anticancer agent it was used [Ru(NO)(bdqi)(terpy)]Cl3 complex as NO delivery agent. The biological effect of that nitrosyl compound and its subproduct after NO release - [Ru(H2O)(bdqi)(terpy)]2+- was evaluated as well as their chemical and photochemical studies. The results lead to the conclusion that NO released from [Ru(NO)(bdqi)(terpy)]3+ has low cytotoxicity effect in B16-F10 cell line but, when it is entrapped in solid lipid nanoparticles, this system is improved and cell viability decreases to 50 %. This effect seems to be dependent on cellular uptake of nitrosyl ruthenium complex. It is also suggested the synergistic activity of the aquoruthenium species once its cell viability decreases to around 25 % after 24 h of incubation with this complex. Synergistic effect of NO and singlet oxygen was also evaluated as a possibility to clinical therapy. Radical oxygen species generation has been used for cancer treatment in clinical therapy known as Photodynamic Therapy (PDT). The success of this therapy depends on oxygen concentration, and hypoxia usually culminates in diminished formation of reactive oxygen species triggering clinical failure of PDT. One of the aims of this thesis is propose synergistic effect of singlet oxygen and NO since it displays antitumor character depending on the NO concentration as an attempt to improve PDT. To this end, the trinuclear species [2RuPc(pz)2](PF6)6 (I) was synthesized and it has been proposed as NO and singlet oxygen photogenerator. Photobiological assays using (I) at 0,4 ?M in B16F10 cell line decreases cell viability to around 30 % under light irradiation at 660 nm, while at the same concentration of compound, without light (I) shows 90 % of cell viability. Studies concerning to cell death mechanism of this compound is also discussed in this work. The potential application of a system like (I) in clinical therapy against cancer must be considered an upgrade for photodynamic therapy.